4-pyridinyl-n-acyl-l-phenylalanines

ABSTRACT

Compounds of Formula I have activity as inhibitors of binding between VCAM-1 and cells expressing VLA-4 and are useful for treating disease whose symptoms and or damage are related to the binding of VCAM-1 to cells expressing VLA-4.

This application is a Continuation of Ser. No. 10/059,618, filed Jan.29, 2002, now abandoned, which claims priority under 35 U.S.C. § 119(e)of Ser. No. 09/717,684, filed Nov. 21, 2000 , now U.S. Pat. No.6,388,084, which is a non-provisional application of Ser. No.60/169,090, filed Dec. 6, 1999, and Ser. No. 60/245,603, filed Nov. 3,2000.

BACKGROUND OF THE INVENTION

Vascular cell adhesion molecule-1 (VCAM-1), a member of theimmunoglobulin (Ig) supergene family, is expressed on activated, but notresting, endothelium. The integrin VLA-4 (α₄β₁), which is expressed onmany cell types including circulating lymphocytes, eosinophils,basophils, and monocytes, but not neutrophils, is the principal receptorfor VCAM-1. Antibodies to VCAM-1 or VLA-4 can block the adhesion ofthese mononuclear leukocytes, as well as melanoma cells, to activatedendothelium in vitro. Antibodies to either protein have been effectiveat inhibiting leukocyte infiltration and preventing tissue damage inseveral animal models of inflammation. Anti-VLA-4 monoclonal antibodieshave been shown to block T-cell emigration in adjuvant-inducedarthritis, prevent eosinophil accumulation and bronchoconstriction inmodels of asthma, and reduce paralysis and inhibit monocyte andlymphocyte infiltration in experimental autoimmune encephalitis (EAE).Anti-VCAM-1 monoclonal antibodies have been shown to prolong thesurvival time of cardiac allografts. Recent studies have demonstratedthat anti-VLA-4 mAbs can prevent insulitis and diabetes in non-obesediabetic mice, and significantly attenuate inflammation in thecotton-top tamarin model of colitis. It has further been shown that VCAMis expressed on endothelial cells of inflamed colonic tissue in aTNB/ethanol rat model of inflammatory bowel disease (Gastroenterology1999, 116, 874-883).

Thus, compounds which inhibit the interaction between α₄-containingintegrins and VCAM-1 will be useful as therapeutic agents for thetreatment of chronic inflammatory diseases such as rheumatoid arthritis(RA), multiple sclerosis (MS), asthma, and inflammatory bowel disease(IBD).

SUMMARY OF THE INVENTION

It has been discovered that compounds of the formula I and thepharmaceutically acceptable salts thereof wherein R₁, R₂, R₃, R₅, R₆ andR₇ are as defined below, inhibit the binding of VCAM-1 to VLA-4 and soare useful in treating inflammatory diseases in which such bindingcontributes to the disease process.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification, the term “halogen” means any of the fourhalogens, bromine, chlorine, fluorine, and iodine unless indicatedotherwise. Preferred halogens are bromine, fluorine, and chlorine.

The term “lower alkyl”, alone or in combination, means a straight-chainor branched-chain alkyl group containing a maximum of six carbon atoms,such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec.butyl,isobutyl, tert.butyl, n-pentyl, n-hexyl and the like. Also, as usedherein “lower alkyl” may be groups which are unsubstituted orsubstituted by one or more groups selected independently fromcycloalkyl, nitro, aryloxy, aryl, hydroxy, halogen, cyano, lower alkoxy,lower alkanoyl, lower alkylthio, lower alkyl sulfinyl, lower alkylsulfonyl, and amino or mono- or di-lower alkyl amino. Examples ofsubstituted lower alkyl groups include 2-hydroxylethyl, 3-oxobutyl,cyanomethyl, and 2-nitropropyl. Although this invention is specificallydirected to the substituted lower alkyl group trifluoromethyl atpositions R₃, R₅, R₂₂ and R₂₃, pentafluoroethyl is also contemplated atthese positions.

The term “cycloalkyl” (or lower cycloalkyl) means an unsubstituted orsubstituted 3- to 7-membered carbacyclic ring. Substituents useful inaccordance with the present invention are hydroxy, halogen, cyano, loweralkoxy, lower alkanoyl, lower alkyl, aroyl, lower alkylthio, lower alkylsulfinyl, lower alkyl sulfonyl, aryl, heteroaryl and substituted amino.

The term “lower alkenyl” means an alkylene group having from 2 to 10carbon atoms with a double bond located between any two adjacent carbonatoms.

The term “lower alkoxy” means a straight-chain or branched-chain alkoxygroup containing a maximum of six carbon atoms, such as methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, tert-butoxy and the like.

The term “lower alkylthio” means a lower alkyl group bonded to the restof the molecule through a divalent sulfur atom, for example, a methylmercapto or a isopropyl mercapto group. The term “lower alkylsulfinyl”means a lower alkyl group as defined above bound to the rest of themolecule through the sulfur atom in the sulfinyl group. The term “loweralkyl sulfonyl” means a lower alkyl group as defined above bound to therest of the molecule through the sulfur atom in the sulfonyl group.

The term “aryl” means a mono- or bicylic aromatic group, such as phenylor naphthyl, which is unsubstituted or substituted by conventionalsubstituent groups. Preferred substituents are lower alkyl, loweralkoxy, hydroxy lower alkyl, hydroxy, hydroxyalkoxy, halogen, loweralkylthio, lower alkylsulfinyl, lower alkylsulfonyl, cyano, nitro,perfluoro lower alkyl, alkanoyl, aroyl, aryl alkynyl, lower alkynyl andlower alkanoylamino. Examples of aryl groups that may be used inaccordance with this invention are phenyl, p-tolyl, p-methoxyphenyl,p-chlorophenyl, m-hydroxy phenyl, m-methylthiophenyl,2-methyl-5-nitrophenyl, 2,6-dichlorophenyl, 1-naphthyl and the like.

The term “arylalkyl” means a lower alkyl group as hereinbefore definedin which one or more hydrogen atoms is/are replaced by an aryl orheteroaryl group as herein defined. Any conventional aralkyl may be usedin accordance with this invention, such as benzyl and the like.

The term “heteroaryl” means an unsubstituted or substituted 5- or6-membered monocyclic hetereoaromatic ring or a 9- or 10-memberedbicyclic hetereoaromatic ring containing 1, 2, 3 or 4 hetereoatoms whichare independently N, S or O. Examples of hetereoaryl rings are pyridine,benzimidazole, indole, imidazole, thiophene, isoquinoline, quinzolineand the like. Substituents as defined above for “aryl” are included inthe definition of heteroaryl.

The term “lower alkoxycarbonyl” means a lower alkoxy group bonded via acarbonyl group. Examples of alkoxycarbonyl groups are ethoxycarbonyl andthe like.

The term “lower alkylcarbonyloxy” means lower alkylcarbonyloxy groupsbonded via an oxygen atom, for example an acetoxy group. This has thesame meaning as the term “acyloxy”.

The term “lower alkanoyl” means lower alkyl groups bonded via a carbonylgroup and embraces in the sense of the foregoing definition groups suchas acetyl, propionyl and the like. The term “perfluoro lower alkanoyl”means a perfluoro lower alkyl group (a substituted lower alkyl groupwhere all of the hydrogens are substituted by fluoro, preferablytrifluoromethyl or pentafluoroethyl) bonded to the rest of the moleculevia a carbonyl group. The term perfluoro lower alkanoylamino” means aperfluoro lower alkanoyl group bonded to the rest of the molecule via anamino group.

The term “lower alkylcarbonylamino” means lower alkylcarbonyl groupsbonded to the rest of the molecule via a nitrogen atom, such asacetylamino. The term lower alkylaminocarbonyl” means lower alkyl aminogroups bonded to the rest of the molecule via a carbonyl group. The term“arylaminocarbonyl” means aryl groups bonded to an amino group furtherbonded to the rest of the molecule via a carbonyl group.

The term “aroyl” means a mono- or bicyclic aryl or heteroaryl groupbonded to the rest of the molecule via a carbonyl group. Examples ofaroyl groups are benzoyl, 3-cyanobenzoyl, 2-naphthoyl, nicotinoyl, andthe like.

Pharmaceutically acceptable salts are well known in the art and can bemade by conventional methods taking into account the chemical nature ofthe compound. Examples of pharmaceutically acceptable salts for acidiccompounds are alkali metal or alkaline earth metals such as sodium,potassium, calcium, magnesium, basic amines or basic amino acids,ammonium or alkyl ammonium salts. Particularly desirable salts forcompounds of this invention are sodium salts. The sodium salt of anyacid of this invention is easily obtained from the acid by treatmentwith sodium hydroxide. For basic compounds, examples are salts ofinorganic or organic acids such as hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, citric, formic, fumaric, maleic, acetic, succinic,tartaric, methanesulfonic, and p-toluenesulfonic.

The present invention comprises a compound of formula I:

and the pharmaceutically acceptable salts thereof.

In accordance with the invention, R₁ is a group Y-1, Y-2 or Y-3 asdescribed below: Y-1 is a group of the formula,

wherein: R₂₂ and R₂₃ are independently hydrogen, lower alkyl, loweralkoxy, cycloalkyl, aryl, arylalkyl, nitro, cyano, lower alkylthio,lower alkylsulfinyl, lower alkyl sulfonyl, lower alkanoyl, halogen, orperfluorolower alkyl and at least one of R₂₂ and R₂₃ is other thanhydrogen, and R₂₄ is hydrogen, lower alkyl, lower alkoxy, aryl, nitro,cyano, lower alkyl sulfonyl, or halogen,

-   Y-2 is a group of the formula Y-2, which is a five or six membered    heteroaromatic ring bonded via a carbon atom to the amide carbonyl    wherein said ring contains one, two or three heteroatoms selected    from the group consisting of N, O and S and one or two atoms of said    ring are independently substituted by lower alkyl, cycloalkyl,    halogen, cyano, perfluoro lower alkyl, or aryl and at least one of    said substituted atoms is adjacent to the carbon atom bonded to the    amide carbonyl;-   Y-3 is a 3-7 membered ring of the formula:    wherein: R₂₅ is lower alkyl, unsubstituted or fluorine substituted    lower alkenyl, or a group of formula R₂₆—(CH₂)_(e)—, R₂₆ is aryl,    heteroaryl, azido, cyano, hydroxy, lower alkoxy, lower    alkoxycarbonyl, lower alkanoyl, lower alkylthio, lower alkyl    sulfonyl, lower alkyl sulfinyl, perfluoro lower alkanoyl, nitro, or    R₂₆ is a group of formula —NR₂₈R₂₉, wherein R₂₈ is hydrogen or lower    alkyl, R₂₉ is hydrogen, lower alkyl, lower alkoxycarbonyl, lower    alkanoyl, aroyl, perfluoro lower alkanoylamino, lower alkyl    sulfonyl, lower alkylaminocarbonyl, arylaminocarbonyl, or R₂₈ and    R₂₉, taken together with the attached nitrogen atom, form a 4, 5 or    6-membered saturated heterocyclic ring optionally containing one    additional heteroatom selected from O, S, and N—R₄₀. Q is    —(CH₂)_(f)O—, —(CH₂)_(f)S—, —(CH₂)_(f)N(R₂₇)—, —(CH₂)_(f)—, R₂₇ is    H, lower alkyl, aryl, lower alkanoyl, aroyl or lower alkoxycarbonyl,    R₄₀ is H, lower alkyl, aryl, lower alkanoyl, aroyl or lower    alkoxycarbonyl, the carbon atoms in the ring are unsubstituted or    substituted by lower alkyl or halogen, e is an integer from 0 to 4,    and f is an integer from 0 to 3;-   R₂ is hydrogen, lower alkyl, substituted lower alkyl, aryl, or aryl    lower alkyl, R₃ is hydrogen, halogen, lower alkyl, or aryl, and may    also include trifluoromethyl, R₄ is hydrogen, halogen, lower alkyl,    or aryl, R₅ is hydrogen, lower alkyl, lower alkoxy, trifluoromethyl,    or OH, R₆ is hydrogen, lower alkyl, lower alkylcarbonyloxy lower    alkyl, or R₆ is a group of formula P-3:    wherein: R₃₂ is hydrogen or lower alkyl R₃₃ is hydrogen, lower    alkyl, aryl, R₃₄ is hydrogen or lower alkyl, h is an integer from 0    to 2, g is an integer from 0 to 2, the sum of h and g is 1 to 3; or    R₆ is a group of formula P-4:    wherein: R₃₂, g, and h are as previously defined, Q′ is O, S,    —(CH₂)_(j)—, or a group of the formula N—R₃₅, R₃₅ is hydrogen, lower    alkyl, lower alkanoyl, lower alkoxycarbonyl, j is 0, 1 or 2, R₇ is    hydrogen, chloro, lower alkoxy, or lower alkyl. Preferably Q is    —(CH₂)_(f)O—, —(CH₂)_(f)S—, or —(CH₂)_(f) (Formula 1a). It is also    preferred in compounds of formula I or Formula 1a that R₅ is    hydrogen, lower alkyl, or trifluoromethyl (Formula 1b).

The compounds of the invention can exist as stereoisomers anddiastereomers, all of which are encompassed within the scope of thepresent invention.

In one preferred embodiment of the compound of formula I, Formula 1a, orFormula 1b, R₂ is hydrogen, lower alkyl, or aryl lower alkyl, R₃ ishydrogen, lower alkyl, or trifluoromethyl, preferably hydrogen, R₄ ishydrogen or halogen, preferably halogen, R₅ is hydrogen, lower alkyl,trifluoromethyl and may in addition be lower alkoxy, but is preferablyhydrogen, R₆ is hydrogen or lower alkyl, and R₇ is hydrogen, chloro,lower alkoxy, or lower alkyl.

A preferred embodiment of the present invention is a compound of theformula I, 1a, or 1b:

wherein R₂₂ and R₂₃ are independently hydrogen, lower alkyl, loweralkoxy, cycloalkyl, aryl, arylalkyl, nitro, cyano, lower alkylthio,lower alkylsulfinyl, lower alkyl sulfonyl, lower alkanoyl, halogen, orperfluorolower alkyl and at least one of R₂₂ and R₂₃ is other thanhydrogen, and R₂₄ is hydrogen, lower alkyl, lower alkoxy, aryl, nitro,cyano, lower alkyl sulfonyl, or halogen. In such compounds, it ispreferred that R₂ is hydrogen, lower alkyl or aryl lower alkyl; R₃ ishydrogen; R₄ is hydrogen, or halogen, or may in addition be lower alkyl(but preferably is halogen); R₅ is hydrogen, lower alkyl, ortrifluoromethyl, or may in addition be lower alkoxy (but preferably ishydrogen); R₆ is hydrogen or lower alkyl; and R₇ is hydrogen, chloro,lower alkoxy, or lower alkyl.

A more preferred embodiment of the present invention is a compound ofthe formula I, Formula 1a, or Formula 1b above wherein R₁ is a group Y-1

wherein R₂₂ and R₂₃ are independently hydrogen, lower alkyl or halogen,especially lower alkyl or halogen; R₂₄ is hydrogen, lower alkyl or loweralkoxy, especially hydrogen. In such compounds, it is preferred that R₂is hydrogen, lower alkyl or aryl lower alkyl; R₃ is hydrogen; R₄ ishydrogen or halogen, R₅ is hydrogen, or lower alkyl, R₆ is hydrogen orlower alkyl; and R₇ is hydrogen, chloro, lower alkoxy, or lower alkyl.In particularly preferred compounds R₂₂ and R₂₃ are independentlyhydrogen, lower alkyl or halogen, R₂₄ is hydrogen or lower alkoxy, andR₂ is or aryl lower alkyl. Alternatively, in such compounds, it ispreferred that R₂ is hydrogen, lower alkyl or aryl lower alkyl; R₃ ishydrogen; R₄ is hydrogen, or halogen, or may in addition be lower alkyl(but preferably is halogen); R₅ is hydrogen, lower alkyl, ortrifluoromethyl, or may in addition be lower alkoxy (but preferably ishydrogen); R₆ is hydrogen or lower alkyl; and R₇ is hydrogen, chloro,lower alkoxy, or lower alkyl.

Another preferred embodiment of the present invention is a compound ofthe formula I, Formula 1a, or Formula 1b above wherein R₁ is a five orsix membered heteroaromatic ring bonded via a carbon atom to the amidecarbonyl wherein said ring contains one, two or three heteroatomsselected from the group consisting of N, O and S and one or two atoms ofsaid ring are independently substituted by lower alkyl, cycloalkyl,halogen, cyano, perfluoro lower alkyl, or aryl and at least one of saidsubstituted atoms is adjacent to the carbon atom bonded to the amidecarbonyl. In such compounds, it is particularly preferred that R₂ ishydrogen, lower alkyl or aryl lower alkyl; R₃ is hydrogen; R₄ ishydrogen or halogen (preferably halogen); R₅ is hydrogen,trifluoromethyl or lower alkyl (preferably hydrogen); R₆ is hydrogen orlower alkyl; and R₇ is hydrogen, chloro, lower alkoxy, or lower alkyl.Alternatively, in such compounds, it is preferred that R₂ is hydrogen,lower alkyl or aryl lower alkyl; R₃ is hydrogen; R₄ is hydrogen, orhalogen, or may in addition be lower alkyl (but preferably is halogen);R₅ is hydrogen, lower alkyl, or trifluoromethyl, or may in addition belower alkoxy (but preferably is hydrogen); R₆ is hydrogen or loweralkyl; and R₇ is hydrogen, chloro, lower alkoxy, or lower alkyl.

Another preferred embodiment of the present invention is a compound ofthe formula I, Formula 1a, or Formula 1b above wherein R₁ is a group ofthe formula Y-3 which is a 3-7 membered ring of the formula:

wherein R₂₅ is lower alkyl, unsubstituted or fluorine substituted loweralkenyl, or a group of formula R₂₆—(CH₂)_(e)—, R₂₆ is aryl, heteroaryl,azido, cyano, hydroxy, lower alkoxy, lower alkoxycarbonyl, loweralkanoyl, lower alkylthio, lower alkyl sulfonyl, lower alkyl sulfinyl,perfluoro lower alkanoyl, nitro, or R₂₆ is a group of formula —NR₂₈R₂₉,wherein R₂₈ is hydrogen or lower alkyl, R₂₉ is hydrogen, lower alkyl,lower alkoxycarbonyl, lower alkanoyl, aroyl, perfluoro loweralkanoylamino, lower alkyl sulfonyl, lower alkylaminocarbonyl,arylaminocarbonyl; or R₂₈ and R₂₉, taken together with the attachednitrogen atom, form a 4, 5 or 6-membered saturated heterocyclic ringoptionally containing one additional heteroatom selected from O, S, andN—R₄₀. Q is —(CH₂)_(f)O—, —(CH₂)_(f)S—, —(CH₂)_(f)N(R₂₇)—, —(CH₂)_(f)—,R₂₇ is H, lower alkyl, aryl, lower alkanoyl, aroyl or loweralkoxycarbonyl, R₄₀ is H, lower alkyl, aryl, lower alkanoyl, aroyl orlower alkoxycarbonyl, the carbon atoms in the ring are unsubstituted orsubstituted by lower alkyl or halogen, e is an integer from 0 to 4, andf is an integer from 0 to 3. In such compounds, it is particularlypreferred that R₂ is hydrogen, lower alkyl or aryl lower alkyl; R₃ ishydrogen; R₄ is hydrogen or halogen (preferably halogen); R₅ ishydrogen, trifluoromethyl, or lower alkyl (preferably hydrogen); R₆ ishydrogen or lower alkyl; and R₇ is hydrogen, chloro, lower alkoxy, orlower alkyl. Alternatively, in such compounds, it is preferred that R₂is hydrogen, lower alkyl or aryl lower alkyl; R₃ is hydrogen; R₄ ishydrogen, or halogen, or may in addition be lower alkyl (but preferablyis halogen); R₅ is hydrogen, lower alkyl, or trifluoromethyl), or may inaddition be lower alkoxy (but preferably is hydrogen); R₆ is hydrogen orlower alkyl; and R₇ is hydrogen, chloro, lower alkoxy, or lower alkyl.

A more preferred embodiment of the present invention is a compound offormula I, Formula 1a, or Formula 1b above wherein R₁ is a group of theformula Y-3 which is a 3-7 membered ring of the formula:

wherein R₂₅ a group of formula R₂₆—(CH₂)_(e)—, wherein R₂₆ is loweralkoxy, Q is —(CH₂)_(f)—, e is an integer from 0 to 4 and f is aninteger from 0 to 3; Such compounds are especially preferred where R2 ishydrogen, lower alkyl, substituted lower alkyl, aryl, or aryl loweralkyl, especially aryl lower alkyl. Such compounds are also preferredwhen R₂ is lower alkyl or aryl lower alkyl; R₃ is hydrogen; R₄ ishydrogen or halogen; R₅ is hydrogen; R₆ is hydrogen or lower alkyl; andR₇ is hydrogen, chloro, lower alkoxy, or lower alkyl. Alternatively, insuch compounds, it is preferred that R₂ is hydrogen, lower alkyl or aryllower alkyl; R₃ is hydrogen; R₄ is hydrogen, or halogen, or may inaddition be lower alkyl (but preferably is halogen); R₅ is hydrogen,lower alkyl, or trifluoromethyl), or may in addition be lower alkoxy(but preferably is hydrogen); R₆ is hydrogen or lower alkyl; and R₇ ishydrogen, chloro, lower alkoxy, or lower alkyl.

In any compound of this invention of formula I, Formula 1a, or Formula1b, R₁ may be a group of formula Y-1 or a group of formula Y-3; R₂ maybe lower alkyl, aryl lower alkyl, or hydrogen (preferably lower alkyl oraryl lower alkyl), R₃ may be hydrogen, lower alkyl, or trifluoromethyl;R₄ may be hydrogen or halogen; R₅ may be hydrogen, lower alkyl, ortrifluoromethyl; R₆ may be hydrogen, lower alkyl, lower alkylcarbonyloxylower alkyl, a group of formula P-3 or a group of formula P-4(preferably the four former groups); and R₇ may be lower alkyl orhydrogen, preferably hydrogen.

In any compound of this invention of formula I, Formula 1a, Formula 1b,or the preferred compounds described in the paragraph immediately above,the groups within Y-1 and Y-3 may be specifically selected as follows:R₂₂ and R₂₃ are hydrogen, halogen, lower alkyl, R₂₄ is hydrogen or loweralkoxy, R₂₅ is a group of formula R₂₆—(CH₂)_(e)—, wherein R₂₆ is loweralkoxy, Q is —(CH₂)_(f)—, e is an integer from 0 to 4 and f is aninteger from 0 to 3, and/or where R₂ is lower alkyl, R₄ is hydrogen, R₃and R₅ are lower alkyl or trifluoromethyl, and R₆ is hydrogen, loweralkyl, or lower alkylcarbonyloxy lower alkyl, or a group of formula P-3preferably where R₃₂ is hydrogen; R₃₃ and R₃₄ are lower alkyl; one of gand h is 1 and the other is 0. In such compounds, preferably R₆ and R₇are hydrogen, and/or R₂ is lower alkyl; R₃ is hydrogen; R₄ is hydrogenor halogen; and R₅ is hydrogen or lower alkyl, and preferably R₂₂ andR₂₃ are lower alkyl or halogen, especially halogen, and R₂₄ is hydrogen,or R₂₂ and R₂₃ are hydrogen or halogen, and R₂₄ is hydrogen or loweralkoxy, preferably hydrogen or R₁ is Y-3. In other such compounds, R₂₂and R₂₃ are preferably halogen, or lower alkyl, R₂₄ is hydrogen, orlower alkoxy, R₂₅ is a group of formula R₂₆—(CH₂)_(e)—, wherein R₂₆ islower alkoxy, Q is —(CH₂)_(f)—, e is an integer from 0 to 4 and f is aninteger from 0 to 3, and/or R₂ is aryl lower alkyl; R₃ is hydrogen, R₄is halogen, and R₅, R₆ and R₇ are hydrogen. It is also preferred that R₁be Y-1.

In a particularly preferred compound of formula I, Formula 1a, orFormula 1b, R₁ is a group of formula Y-1 or a group of formula Y-3; R₂is lower alkyl or aryl lower alkyl; R₃ is hydrogen, lower alkyl, ortrifluoromethyl; R₄ is hydrogen or halogen; R₅ is hydrogen, lower alkyl,or trifluoromethyl; R₆ is hydrogen, lower alkyl, lower alkylcarbonyloxylower alkyl, or a group of formula P-3; and R₇ is hydrogen (Formula A)

In a preferred compound of Formula A, the groups within Y-1 and Y-3 maybe specifically selected as follows: R₂₂ and R₂₃ are hydrogen, halogen,or lower alkyl, R₂₄ is hydrogen, or lower alkoxy, R₂₅ is a group offormula R₂₆—(CH₂)_(e)—, wherein R₂₆ is lower alkoxy, Q is —(CH₂)_(f)—, eis an integer from 0 to 4 and f is an integer from 0 to 3 (Formula A-1).

In a preferred compound of Formula A-1, R₂ is lower alkyl, R₄ ishydrogen, and R₃ and R₅ are lower alkyl or trifluoromethyl (FormulaA-1a). In one embodiment of Formula A-1a, R₆ is hydrogen. In thisembodiment, R₁ may be Y-1, especially where R₂₂ and R₂₃ are halogen orlower alkyl, or R₁ may be Y-3. In other embodiments of Formula A-1 a, R₆is lower alkyl, or is lower alkylcarbonyloxy lower alkyl, or is a groupof formula P-3 wherein R₃₂ is hydrogen; R₃₃ and R₃₄ are lower alkyl; oneof g and h is 1 and the other is 0.

In another preferred compound of Formula A-1, R₆ is hydrogen. It ispreferred that R₂ is lower alkyl; R₃ is hydrogen; R₄ is hydrogen orhalogen; and R₅ is hydrogen or lower alkyl (Formula A-1b). In oneembodiment of Formula A-1b, R₁ is Y-1. Preferably R₂₂ and R₂₃ are loweralkyl or halogen, and R₂₄ is hydrogen, or R₂₂ and R₂₃ are halogen, andR₂₄ is hydrogen, or R₂₂ and R₂₄ are hydrogen or halogen, and R₂₃ islower alkoxy. In another embodiment of Formula A-1b, R₁ is Y-3.

In another preferred compound of Formula A, R₂₂ and R₂₃ are hydrogen,halogen, or lower alkyl, R₂₄ is hydrogen or lower alkoxy, R₂₅ is a groupof formula R₂₆—(CH₂)_(e)—, wherein R₂₆ is lower alkoxy, Q is—(CH₂)_(f)—, e is an integer from 0 to 4 and f is an integer from 0 to3. Preferably R₂ is aryl lower alkyl; R₃ is hydrogen, R₄ is halogen, andR₅, R₆ and R₇ are hydrogen. It is also preferred that R₁ be Y-1 or Y-3.

The compounds of the invention inhibit the binding of VCAM-1 andfibronectin to VLA-4 on circulating lymphocytes, eosinophils, basophils,and monocytes (“VLA-4-expressing cells”). The binding of VCAM-1 andfibronectin to VLA-4 on such cells is known to be implicated in certaindisease states, such as rheumatoid arthritis, multiple sclerosis,inflammatory bowel disease, and particularly in the binding ofeosinophils to airway endothelium which contributes to the cause of thelung inflammation which occurs in asthma. Thus, the compounds of thepresent invention would be useful for the treatment of asthma.

On the basis of their capability of inhibiting binding of VCAM-1 andfibronectin to VLA-4 on circulating lymphocytes, eosinophils, basophils,and monocytes, the compounds of the invention can be used as medicamentfor the treatment of disorders which are known to be associated withsuch binding. Examples of such disorders are rheumatoid arthritis,multiple sclerosis, asthma, and inflammatory bowel disease. Thecompounds of the invention are preferably used in the treatment ofdiseases which involve pulmonary inflammation, such as asthma. Thepulmonary inflammation which occurs in asthma is related to theactivation and lung infiltration of eosinophils, monocytes andlymphocytes which have been activated by some asthma-triggering event orsubstance.

Furthermore, compounds of the invention also inhibit the binding ofMadCAM to the cellular receptor alpha4-beta7, also known as LPAM, whichis expressed on lymphocytes, eosinophiles and T-cells. While the preciserole of alpha4-beta7 interaction with various ligands in inflammatoryconditions such as asthma is not completely understood, compounds of theinvention which inhibit both alpha4-beta1 and alpha4-beta7 receptorbinding are particularly effective in animal models of asthma.Furthermore work with monoclonal antibodies to alpha4-beta7 indicatethat compounds which inhibit alpha4-beta7 binding to MadCAM are usefulfor the treatment of inflammatory bowel disease. They would also beuseful in the treatment of other diseases in which such binding isimplicated as a cause of disease damage or symptoms.

The compounds of the invention can be administered orally, rectally, orparentally, e.g., intravenously, intramuscularly, subcutaneously,intrathecally or transdermally; or sublingually, or as opthalmalogicalpreparations, or as an aerosol in the case of pulmonary inflammation.Capsules, tablets, suspensions or solutions for oral administration,suppositories, injection solutions, eye drops, salves or spray solutionsare examples of administration forms.

Intravenous, intramuscular, oral or inhalation administration is apreferred form of use. The dosages in which the compounds of theinvention are administered in effective amounts depending on the natureof the specific active ingredient, the age and the requirements of thepatient and the mode of administration. Dosages may be determined by anyconventional means, e.g., by dose-limiting clinical trials. Thus, theinvention further comprises a method of treating a host suffering from adisease in which VCAM-1 or fibronectin binding to VLA-4-expressing cellsis a causative factor in the disease symptoms or damage by administeringan amount of a compound of the invention sufficient to inhibit VCAM-1 orfibronectin binding to VLA-4-expressing cells so that said symptoms orsaid damage is reduced. In general, dosages of about 0.1-100 mg/kg bodyweight per day are preferred, with dosages of 1-25 mg/kg per day beingparticularly preferred, and dosages of 1-10 mg/kg body weight per daybeing espeically preferred.

The invention further comprises pharmaceutical compositions whichcontain a pharmaceutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier. Such compositionsmay be formulated by any conventional means. Tablets or granulates cancontain a series of binders, fillers, carriers or diluents. Liquidcompositions can be, for example, in the form of a sterilewater-miscible solution. Capsules can contain a filler or thickener inaddition to the active ingredient. Furthermore, flavour-improvingadditives as well as substances usually used as preserving, stabilizing,moisture-retaining and emulsifying agents as well as salts for varyingthe osmotic pressure, buffers and other additives can also be present.

The previously mentioned carrier materials and diluents can comprise anyconventional pharmaceutically acceptable organic or inorganicsubstances, e.g., water, gelatine, lactose, starch, magnesium stearate,talc, gum arabic, polyalkylene glycols and the like.

Oral unit dosage forms, such as tablets and capsules, preferably containfrom 25 mg to 1000 mg of a compound of the invention.

Generally the compounds of the present invention can be prepared fromsuitable phenylalanine derivatives via a palladium catalyzed reactionwith a 3-halo-2-pyridone.

As shown in Reaction Scheme 1, a 4-iodo- or 4-bromophenylalaninederivative such as 1, is converted into a protected phenylalaninederivative 2 in which R₇′ is hydrogen, chloro, lower alkyl or loweralkoxy, P₁ is a standard nitrogen protecting group such as a Boc, orcarbobenzyloxy group and P₂ is lower alkyl or substituted lower alkylselected appropriately to serve as a protecting group or an element of aprodrug. The group P₂ can be introduced by conventional means familiarto those who practice peptide chemistry. The order of the addition of P₁and P₂ is not critical and will depend on the particular choice ofreagents. A discussion of the use and introduction of protecting groupsis provided in Theodora W. Greene and Peter G. M. Wuts., ProtectingGroups in Organic Synthesis, Wiley Interscience, New York, 1991.Alternatively, a compound of formula 1 may be converted to a compound offormula 4, in which R₁′ represents a component of an acyl group of theinvention. A convenient method is to introduce the ester group P₂ first,followed by a coupling reaction of the free amine using conventionalpeptide coupling reagents, for example HBTU in the presence of atertiary amine base such as diethylisopropylamine. Again, the particularchoice of reagents may dictate altering the sequence of the introductionof R₁′ and P₂. Conversion of compounds of formula 2 or 4 to derivatives3 or 5, in which M represents a substituted tin or boron atom, can beeffected by treatment with a suitable species, for examplehexamethylditin, hexabutylditin or a tetraalkoxydiboron in the presenceof a source of palladium zero. The methodology is outlined andreferenced in F. Diederich and P. J. Stang, ed, Metal Catalyzed CrossCoupling Reactions, Wiley-VCH, Weinheim, Germany, 1998.

Substituted pyridones of formula 6 wherein R_(3′), R_(4′), and R_(5′)are hydrogen, lower alkyl, perfluorolower alkyl, halogen, aryl, hydroxy,or lower alkoxy are commercially available or prepared following generalliterature methods (for example, discussion of some of these knownmethods are found in: P. Wirth, et al., Ger. Offen. 28 17 293, Aug. 12,1979, Masaru Hojo, et al. Chem. Lett. 1976, 499-502, Robert W. Lang, etal. Helvetica Chimica Acta, 1988, 71, 596-601). Compounds of formula 7wherein R_(2′) is lower alkyl or aryl lower alkyl, may be obtained byalkylation of compounds of formula 6 by treatment with an alkylatingagent such as iodomethane, benzylbromide in the presence of a base suchas potassium carbonate and optionally, a phase transfer catalyst. Forless reactive alkylating agents, it may be necessary to use a strongerbase such as an alkali metal hydroxide and to heat the reaction mixture.Compounds of formula 7 as defined above may be halogenated in the3-position by treatment with conventional halogenating reagents such asbromine, N-iodosuccinimide or N-bromosuccinimide in a suitable solventsuch as glacial acetic acid or aqueous acetic acid to give halopyridonesof formula 8, X=Br or I, R_(2′) is independently hydrogen, lower alkyl,substituted lower alkyl, or aryl lower alkyl, R_(3′) and R_(5′) arehydrogen, lower alkyl or perfluorolower alkyl. Treatment with excesshalogenating agent leads to compounds of structure 8 withR_(4′)=halogen. For compounds of formula 8 with R_(2′)=aryl, it isconvenient to start with N-aryl pyridones which are known compounds orcan be prepared by known methods, or to introduce an aryl moiety forexample by using an Ullmann coupling reaction as described by Masakatsu,et al., Chem. Pharm. Bull. 1997, 45, 719.

As shown in Reaction Scheme 3, a compound of formula 8 can be used in apalladium catalyzed coupling reaction with a phenylalanine derivative offormula 3 or 5. For example, when M is a substituted tin, treatment of amixture of 8 and the phenylalanine of formula 3 or 5 with a source ofpalladium zero such as tetraakis(triphenylphosphine)palladium orbis(triphenylphosphine)palladium dichloride in the presence of an inertsolvent such as DMF at a temperature of between room temperature and100° C. gives a compound of formula 9 or 10. Compounds of structure 9may be converted into compounds of structure 10 by removal of theprotecting group P₁, which may be accomplished by conventional meansdepending on the selection of P₁. For example, if P₁ is a Boc group, itmay be removed by treatment with a strong acid, such as trifluoroaceticacid, optionally in the presence of a solvent such as methylene chlorideand a scavenging agent. The resulting free amine may then be acylatedwith an acid of the formula R₁′CO₂H using convention peptide couplingtechniques. For example, by treatment with HBTU in the presence of atertiary amine base such as diethylisopropylamine in the presence of anaprotic solvent such as DMF to give the compound of structure 10.

If the free acid 11 is the desired end product, the ester group, P₂ maybe removed by conventional means. For example, in the case that P₂ islower alkyl, for example methyl, it may be removed by treatment with analkali metal hydroxide, for example lithium hydroxide, in a suitablesolvent, for example aqueous THF optionally containing methanol toassist with solubility. If P₂ were a benzyl or substituted benzyl group,it could also be removed by catalytic hydrogenation over a noble metalcatalyst, for example palladium on carbon.

Alternatively, as shown in Reaction Scheme 4, a compound of structure 8,wherein X is bromide or iodide, may be converted to a species of formula12, in which M′ represents a substituted tin, boron or zinc atom. In thecase of the tin or boron derivatives, in which M′ represents asubstituted tin or boron atom, the conversion can be effected bytreatment with a suitable species, for

example hexamethylditin, hexabutylditin or a tetraalkoxydiboron in thepresence of a source of palladium zero. For the formation of the zincderivative, 12, M′=Zn(halogen), conversion may be effected by treatmentof the compound of formula 8, X=I with a source of activated zinc metalin a suitable inert solvent, for example dimethylacetamide at atemperature of from room temperature to 100° C. until conversion iscomplete to give a compound of formula 12, M′=Zn(halogen). Thesecompounds of formula 12 can be reacted with a 4-substitutedphenylalanine derivative of formula 4′ in which X′ is iodo, bromo, ortrifluoromethylsulfonyloxy in the presence of a source of palladium zeroto give a compound of formula 10′. In the case where the ester grouprepresented by P₂ is not part of the targeted compound, it can beremoved using ester hydrolysis procedures appropriate to the particularP₂. For example, where P₂ is lower alkyl, for example methyl, it can beremoved by standard base hydrolysis using an alkali metal hydroxide, forexample, lithium hydroxide. In a variation on this procedure, it may bedesirable to carry a protecting group through the coupling reaction andsubstitute it at a later time. In this case, a compound of formula 2′,in which P₁′ is lower alkoxycarbonyl or benzyloxycarbonyl and X′ is asdefined above, may be coupled with a pyridone of structure 12 to give acompound of structure 9′ which in turn may be converted to a compound ofthe invention using the general procedures noted above in reactionscheme 3.

An alternative route to compounds of this invention, as shown inReaction Scheme 5, which is particularly applicable to compounds inwhich R₇′ is other than hydrogen, is to build an aldedyde of formula 14.This can be accomplished by reacting a compound of formula 12 with acompound of formula 13, in which R₇′ represents lower alkyl or loweralkoxy, and X″ represents an iodide, bromide, oftrifluoromethylsulfonyloxy moiety and R₈ represents a protected alcoholor an aldehyde. For alcohols, suitable protecting groups include silylethers, benzyl ethers. If necessary, aldehydes, may be protected astheir acetal derivatives. The compound of formula 12 can be converted toan aldehyde of formula 15 by convertional steps which, when R₈ is analcohol, would involve protecting group removal, if necessary, followedby oxidation. Any of the common reagents for the selective oxidation ofprimary benzyl alcohols to aldehydes may be employed, for example,treatment with activated manganese dioxide in an inert solvent. In thecase where R₈ represents a protected aldehyde, conversion to an aldehydeof formula 15 can be carried out by a suitable protecting group removal,for example hydrolysis of an acetal with dilute acid. Reaction of 15 togive a dehydroamino acid of formula 16 can be effected by treatment witha Wittig reagent of formula 17 in which P₁′ is lower alkoxycarbonyl orbenzyloxycarbonyl and P₂ is as defined above. For example treatment of15 with (±)-N-(benzyloxycarbonyl)-α-phosphonoglycine trimethyl ester inthe presence of a suitable base for example tetramethylguanidine leadsdirectly to a dehydroamino acid of formula 16, P₂=methyl andP_(1′)=benzyloxycarbonyl. Enantioselective reduction of 16 to theL-amino acid 18 can be effected by use of a number of reducing agentssuitable for the purpose, for example, the recently describedethyl-DuPHOS rhodium reagent (Burk, M. J., Feaster, J. E.; Nugent, W.A.; Harlow, R. L. J. Am. Chem. Soc. 1993, 115, 10125) using essentiallythe literature procedure. Further conversion of 18 to the compounds ofthe invention can be carried out using the general procedures discussedabove. Alternatively, the general methods outlined in reaction scheme 5can be used to prepare compounds of structure 4 or 4′ in which R_(7′) isother than hydrogen. These compounds of structure 4 or 4′ can then beemployed to prepare compounds of the invention as outlined in reactionschemes 1 to 4.

In one embodiment, the N-acyl group, R₁′ of structure 11, is derivedfrom a 2-subsituted benzoic acid. Appropriate 2-substituted benzoicacids are either commercially available or can be prepared byconventional means. For example ortho-substituted aryl iodides ortriflates may be carbonylated in the presence of carbon monoxide and asuitable palladium catalyst. The preparation of such iodide or triflateintermediates is dependent on the particular substitution patterndesired and they may be obtained by direct iodination or diazotizationof an aniline followed by treatment with a source of iodide for example,potassium iodide. Triflates may be derived from the correspondingphenols by conventional means such as by treatment with trifluoromethanesulfonic anhydride in the presence of a base such as triethylamine ordiisopropylethylamine in an inert solvent. As shown in Reaction Scheme6, one other means of obtaining ortho-substituted benzoic acids involvestreatment of an 2-methoxyphenyloxazoline derivative such as compound 19,Z₁ and Z₂=hydrogen, alkyl, chloro, perfluoro lower alkyl, lower alkoxywith an alkyl Grignard reagent followed by hydrolysis of the oxazolinering following the general procedure described by Meyers, A. I., Gabel,R., Mihelick, E. D, J. Org. Chem. 1978, 43, 1372-1379, to give an acidof formula 20. 2- or 2,6-Disubstituted benzonitriles also serve asconvenient precursors to the corresponding benzoic acids. In the case ofhighly hindered nitrites, for example 2-chloro-6-methylbenzonitrile,conventional hydrolysis under acidic or basic conditions is difficultand better results are obtained by DIBAL reduction to the correspondingbenzaldehyde followed by oxidation using a chromium based oxidizingreagent. Other methods are exemplified in Chen, et al., WO 9910312.

Referring now to Reaction Scheme 7, cyclic acids of formula 23 are knowncompounds or can be prepared using standard methodologies. For thepreparation of substituted alkyl- or cycloalkylcarboxylic acids,alkylation reactions can be employed using an alkali metal dianion ofthe acid or monoanion of the corresponding ester. For example, acycloalkyl carboxylic acid ester of formula 21 can be treated with astrong base, for example, lithium diisopropylamide in an inert solvent,for example THF followed by addition of group R₄₁-Lv wherein R₄₁represents a desired side chain, such as a substituted benzyl, loweralkyl, lower alkoxy alkyl, azido lower alkyl and the like and Lvrepresents a leaving group such as a bromide, iodide, mesylate orsimilar group known to participate in ester enolate alkylationreactions. The product ester 22 may be hydrolyzed to the acid 23 usingalkali metal hydroxide in a suitable solvent, for example aqueousalcohol. Depending on the nature of R₄₁ and the eventual target, thecompound 23 may be coupled to an amine such as compound 1 and convertedto the target directly or R₄₁ may be subject to further manipulation ata suitable point in the synthesis. For example, if R₄₁ is an azido loweralkyl moiety, the azide may be reduced using for example a trialkylphosphine reagent followed by functionalization of the product amine byalkylation, acylation, sulfonylation and related procedures well knownto those skilled in the art. If R⁴¹ incorporates a leaving group, forexample, a terminal bromine atom, this group may be displaced by anappropriate nucleophile, for example, sodium methyl mercaptide to givein this case, a thioether which may be the desired product or can beitself further manipulated, for example by oxidation to a sulfoxide orsulfone using standard reaction conditions. Other nucleophiles which maybe employed to produce intermediates leading to compounds of thisinvention include: sodium cyanide, sodium methoxide, sodium azide,morpholine and others. When R₄₁ incorporates a ketal group, this groupmay be hydrolzyed at a convenient point in the synthesis to provide aketo group. This group in turn may be further manipulated, for exampleby reduction to an alcohol or conversion to derivative such as an oxime.

Examples of the application of these methods to the synthesis ofcompounds of formula 23 are provided in Chen, et al. WO 9910313.

In general, ortho-substituted aromatic acids needed for the preparationof compounds in which R¹=Y-1 can be prepared as exemplified in Chen, etal., WO9910312.

For the synthesis of 2-chloro-6-alkylbenzoic acids of formula 28,wherein R₄₃ is lower alkyl or cycloalkyl, the procedure described inReaction Scheme 8 is particularly suitable. Thus a commerciallyavailable aldehyde of formula 24 is converted to the imine 25 whereinR42 is lower alkyl, preferably butyl, by treatment with butylamine in aninert, hydrophobic organic solvent, for example heptane. The resultingcompound of formula 25 is treated with an excess of a Grignardderivative 26 in an inert solvent, for example THF, followed by acidtreatment during the workup to give an aldehyde of formula 27. Oxidationof 27 to an acid of formula 28 can be carried out by conventional means,for example by treatment of a solution of 27 in a suitable solvent suchas aqueous acetonitrile with sodium chlorite and 30% hydrogen peroxideat or below room temperature.

It may be desirable to prepare prodrug esters of the compounds of thisinvention for which it would be more convenient to introduce the estermoiety at the end of the synthesis. For this purpose, a variety ofcommon techniques for the formation of esters from carboxylic acids maybe employed. Typical methods which may be useful would include, couplingof an alcohol to the carboxylic acid in the presence of acid, forexample hydrochloric acid, a procedure commonly known as a Fisheresterification. Alternatively, a diimide mediated coupling between thecarboxylic acid and an alcohol may be employed with the optional use ofa promoter such as 4,4-dimethylaminopyridine. A typical diimide isdicyclohexylcarbodiimide. Another alternative is to treat the carboxylicacid with a reactive alkyl halide, for example, an alkyl iodide or anacyloxymethyl chloride in the presence of a base, for example sodiumbicarbonate and an inert solvent, for example DMF. The particular choiceof method will be determined by the nature of the particular combinationof carboxylic acid and desired ester moiety and will be apparent to oneskilled in the art. Ester groups which may constitute prodrugs may beintroduced at any convenient point in the synthesis. For example thegroup P₂ in formula 1 may represent a desirable prodrug ester and beretained in the final product.

EXAMPLES

The Examples which follow are for purposes of illustration and are notintended to limit the invention in any way.

General Methods: Melting points were taken on a Thomas-Hoover apparatusand are uncorrected. Optical rotations were determined with aPerkin-Elmer model 241 polarimeter. ¹H-NMR spectra were recorded withVarian XL-200, Mercury 300 and Unityplus 400 MHz spectrometers, usingtetramethylsilane (TMS) as internal standard. Electron impact (EI, 70ev) and fast atom bombardment (FAB) mass spectra were taken on VGAutospec or VG 70E-HF mass spectrometers. Silica gel used for columnchromatography was Mallinkrodt SiliCar 230-400 mesh silica gel for flashchromatography; columns were run under a 0-5 psi head of nitrogen toassist flow. Thin layer chromatograms were run on glass thin layerplates coated with silica gel as supplied by E. Merck (E. Merck #1.05719) and were visualized by viewing under 254 nm UV light in a viewbox, by exposure to I₂ vapor, or by spraying with either phosphomolybdicacid (PMA) in aqueous ethanol, or after exposure to Cl₂, with a4,4′-tetramethyldiaminodiphenylmethane reagent prepared according to E.Von Arx, M. Faupel and M Brugger, J. Chromatography, 1976, 120, 224-228.

Reversed phase high pressure liquid chromatography (RP-HPLC) was carriedout using a Rainin HPLC employing a 41.4×300 mm, 8 μM, Dynamax™ C-18column at a flow of 49 mL/min employing a gradient of acetonitrile:water(each containing 0.75% TFA) typically from 5 to 95% acetonitrile over35-40 min. HPLC conditions are typically described in the format(5-95-35-214); this refers to a linear gradient of from 5% to 95%acetonitrile in water over 35 min while monitoring the effluent with aUV detector at a wavelength of 214 nM.

Methylene chloride (dichloromethane), 2-propanol, DMF, THF, toluene,hexane, ether, and methanol, were Fisher reagent grade and were usedwithout additional purification except as noted, acetonitrile was Fisheror Baker hplc grade and was used as is.

Definitions as used herein:

-   THF is tetrahydrofuran,-   DMF is N,N-dimethylformamide,-   DMA is N,N-dimethylacetamide-   HOBT is 1-hydroxybenzotriazole,-   BOP is [(benzotriazole-1-yl)oxy]tris-(dimethylamino)phosphonium    hexafluorophosphate,-   HATU is O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HBTU is O-benzotriazole-N,N,N′,N′,-tetramethyluronium    hexafluorophosphate,-   DIPEA is diisopropylethylamine,-   DMAP is 4-(N,N-dimethylamino)pyridine-   DPPA is diphenylphosphoryl azide-   DPPP is 1,3-bis(diphenylphosphino)propane-   DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene-   NaH is sodium hydride-   brine is saturated aqueous sodium chloride solution-   TLC is thin layer chromatography-   LDA is lithium diisopropylamide-   BOP-Cl is bis(2-oxo-3-oxazolidinyl)phosphinic chloride-   NMP is N-methyl pyrrolidinone-   Lawesson's reagent is    [2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide]-   NIS is N-iodosuccinimide.    Silica gel chromatography on Biotage columns refers to use of a    flash chromatography system supplied by the Biotage Division of the    Dyax Corporation employing prepacked 40 g (40 s columns), 90 g (40 m    columns) or 800 g (75 m columns). Elution is carried out with    hexane-ethyl acetate mixtures under 10-15 psi nitrogen pressure.

Example 1N-[(1,1-dimethylethoxy)carbonyl]-4-[(tributyl)stannyl]-L-phenylalaninemethyl ester

A solution of N-[(1,1-dimethylethoxy)carbonyl]-4-iodo-L-phenylalaninemethyl ester (5.3 g, 13 mmol) and hexabutylditin (27.5 mL, 54 mmol) intoluene (50 mL) was deoxygenated by alternately freezing the mixture ina liquid nitrogen bath under vacuum and thawing under argon (3×).

Tetrakis(triphenylphosphine)palladium was added (280 mg, 0.22 mmol) andthe reaction mixture was heated to reflux for 45 min as the colorchanged from yellow to black. TLC (1:6 ethyl acetate:hexane) indicatedthe presence of some starting iodide and an additional portion (140 mg,0.11 mmol) of the catalyst was added. Reflux was continued for 1 hr. Themixture was allowed to cool and was concentrated. The residue was takenup in hexane (200 mL) and triethylamine (30 mL), stirred for 30 min andwas filtered. The filtrate was concentrated and was chromatographed overa dry silica gel column containing 150 g of silica gel and eluting withhexane followed by 1:6 ethyl acetate:hexanes to giveN-[(1,1-dimethylethoxy)carbonyl]-4-[(tributyl)stannyl]-L-phenylalaninemethyl ester (5.7 g, 77%) as a clear oil. LR(+)LSIMS (C27H47NO4Sn): m/z1081 (2M-C4H9) 570 (M+H).

Example 2 2-Amino-3-bromo-5-chloropyridine

A suspension of 2-amino-5-chloropyridine (5.14 g, 40 mmol) and anhydroussodium acetate (3.29 g, 40 mmol) in acetic acid (25 mL) was mechanicallystirred and warmed to a bath temperature of 45° C. A solution of bromine(2.1 mL, 40 mmol) in acetic acid (2 mL) was added over 1 hour. Theresulting orange mixture was cooled to 15° C. and filtered. The solidswere taken up in 400 mL of water, the suspension made basic by additionof 1 N NaOH and the suspension extracted with ethyl acetate (5×100 mL).The combined extracts were washed with 10% NaHSO₃ (1×100 mL) and weredried over MgSO₄. Concentration afforded2-amino-3-bromo-5-chloropyridine (4 g, 48), mp 82-84° C.

Example 3

A solution of 2-amino-3-bromo-5-chloropyridine (4.0 g, 19.3 mmol) inwater (30 mL) and conc. HCl (5.2 mL) was cooled with an ice bath to 0°C. and was treated with a solution of sodium nitrite (1.33 g, 19.3 mmol)in water (12 mL) over 10 min. The mixture was stirred for an additonal10 min and allowed to warm to room temperature over 48 hr. The mixturewas filtered. The solids were washed with water, then with CCl₄ and weredried under vacuum at 50° C. for 3 hr to give3-bromo-5-chloro-1H-2-pyridone (2.77 g, 69%), mp 173.5-175° C.

Example 4

A mixture of 3-bromo-5-chloro-1H-2-pyridone (930 mg, 4.46 mmol),potassium carbonate (1.25 g, 9.1 mmol) and iodomethane (2.8 mL, 45 mmol)in 10 mL of DME was heated to reflux for 18 hr. The mixture was filteredhot and concentrated. The residue was recrystallized from ethyl acetateto give 3-bromo-5-chloro-1-methyl-2-pyridone (740 mg, 74%), mp 162-163°C. HRMS (C6H5BrClNO): Obs. Mass 220.9249. Calcd. Mass 220.9243 (M+H).

Example 54-(5-Chloro-1-methyl-2-oxo-3-pyridinyl)-N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalaninemethyl ester

A solution of 3-bromo-5-chloro-1-methyl-2-pyridone (660 mg, 2.97 mmol)andN-[(1,1-dimethylethoxy)carbonyl]-4-[(tributyl)stannyl]-L-phenylalaninemethyl ester (1.7 g, 2.99 mmol) in DMF (30 mL) was deoxygenated byalternately freezing the mixture in a liquid nitrogen bath under vacuumand thawing under argon (3×). Tetrakis(triphenylphosphine)palladium (140mg, 0.20 mmol) was added and the mixture was heated to 90° C. for 3 hras the mixture turned dark. TLC indicated that the reaction was notcomplete and an additional 140 mg portion of the catalyst was added andheating continued for 4 hr. The mixture was allowed to cool and wasdiluted with dichloromethane and was filtered through a pad of celite.The filtrate was evaporated to dryness and the residue was dissolved in1:1 ether:ethyl acetate (60 mL). The solution was washed with water(3×10 mL) and brine (1×10 mL) and was dried (MgSO₄). The residueobtained upon concentration was chromatographed over 150 g of silica geleluting with 7:3 ethyl acetate:hexane to give4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalaninemethyl ester (670 mg, 54%). LRMS-Electrospray: m/z 863 (2M+Na), 858(2M+NH4), 443 (M+Na), 438 (M+NH4), 421(M+H).

Example 6 4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine methylester hydrochloride

A solution of4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalaninemethyl ester (210 mg, 0.50 mmol) in 4 N HCl in dioxane (10 mL) wasstirred for 1 hr and was concentrated to give4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine methyl ester(140 mg) as a white powder. LR(+)LSIMS: m/z 641 (2M+H), 321 (M+H).

Example 7N-[(2-Chloro-6-methylphenyl)carbonyl]-4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester

A solution of 4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester hydrochloride (89.5 mg, 0.25 mmol),2-chloro-6-methylbenzoic acid (47 mg, 0.28 mmol), DIEA (175 μL, 1.0mmol) and HBTU (133 mg, 0.35 mmol) in DMF (3 mL) was stirred at roomtemperature for 4 hr. The mixture was concentrated, the residue wasdissolved in ethyl acetate (15 mL), was washed with 0.5 N HCl (1×5 mL),sat. NaHCO₃ (1×5 mL), brine (1×5 mL) and was dried (MgSO₄). The residueobtained upon evaporation was chromatographed on 25 g of silica gel,eluting with 3:1 ethyl acetate:hexane to giveN-[(2-chloro-6-methylphenyl)carbonyl]-4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (54 mg). LR(+)LSIMS: m/z 962 (2M+NH4), 945 (2M+H), 490(M+NH4), 473 (M+H (2 Cl)).

Example 8N-[(2-chloro-6-methylphenyl)carbonyl]-4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine

A solution ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (51 mg, 0.108 mmol) in THF (3 mL) was treated with asolution of lithium hydroxide monohydrate (18 mg, 0.43 mmol) in water(1.0 mL). Additional THF was added to effect a clear solution and thereaction mixture was stirred 1 hr. TLC (ethyl acetate) indicatedstarting material was consumed. A few drops of acetic were added and theentire reaction mixture was applied to a 4×30 cm, C-18 reversed phaseHPLC column and eluted with a linear gradient of acetonitrile in waterof 5 to 95% over 35 min. The product containing fraction waslyophillyzed to giveN-[(2-chloro-6-methylphenyl)carbonyl]-4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine(40 mg, 82%) as a white solid. LR-Electrospray: m/z positive ion,481(M+Na), 459(M+H (2 Cl)); negative ion, 457 (M−H (2 Cl)).

Example 94-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalaninemethyl ester

A solution of 4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester hydrochloride (47.4 mg, 0.25 mmol),1-(2-methoxyethyl)cyclopentane carboxylic acid (47 mg, 0.28 mmol), DIEA(175 μL, 1.0 mmol) and HBTU (133 mg, 0.35 mmol) in DMF (3 mL) wasstirred at room temperature for 4 hr. The mixture was concentrated, theresidue was dissolved in ethyl acetate (15 mL), was washed with 0.5 NHCl (1×5 mL), sat. NaHCO₃ (1×5 mL), brine (1×5 mL) and was dried(MgSO₄). The residue obtained upon evaporation was chromatographed on 25g of silica gel, eluting with ethyl acetate to give4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalaninemethyl ester (70.5 mg, 59%). LR-Electrospray: m/z positive ion, 943(2M+Na), 483 (M+Na), 478, (M+NH4), 461 (M+H); negative ion 919 (2M-H),521, 459 (M−H).

Example 104-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine

A solution of4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalaninemethyl ester (69 mg, 0.145 mmol) in THF (3 mL) was treated with asolution of lithium hydroxide monohydrate (24.3 mg, 0.58 mmol) in water(1.0 mL). Sufficient THF was added to the mixture to effect solution.The mixture was stirred 1 hr and a few drops of acetic acid were added.The entire reaction mixture was applied to a 4×30 cm, C-18 reversedphase HPLC column and eluted with a linear gradient of acetonitrile inwater of 5 to 95% over 35 min. The product containing fraction waslyophillyzed to give4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine(48 mg, 72%) as a white solid. LR-Electrospray: m/z positive ion 943(2M+Na), 483 (M+Na), 478, (M+NH4), 461 (M+H); negative ion 919 (2M−H),521, 459 (M−H).

Example 11 Benzyl-3-bromo-5-chloro-2-pyridone

A mixture of 3-bromo-5-chloro-1H-2-pyridone (950 mg, 4.56 mmol), freshlyground potassium carbonate (1.26 g, 9.1 mmol) and tetrabutyl ammoniumchloride (100 mg) in was stirred mechanically without solvent for 30 minand benzyl bromide (0.56 mL, 4.7 mmol) was added. The mixture wasstirred at room temperature for 72 hr. The mixture was diluted withethyl acetate and filtered, washing the solids with ethyl acetate. Thefiltrate was evaporated to dryness and the residue was triturated withseveral portions of hexane. The residue was chromatographed over 90 g ofsilica gel eluting with 3:1 hexane:ethyl acetate followed by 100% ethylacetate to give 1-benzyl-3-bromo-5-chloro-2-pyridone (423 mg, 31%), mp108-110° C. LR(+)LSIMS: m/z 595 (2M+H, 2 Cl, 2 Br), 298 (M+H, 1 Cl, 1Br).

Example 124-(1-Benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalaninemethyl ester

A solution of 1-benzyl-3-bromo-5-chloro-2-pyridone (296 mg, 0.98 mmol)andN-[(1,1-dimethylethoxy)carbonyl]-4-[(tributyl)stannyl]-L-phenylalaninemethyl ester (560 mg, 0.99 mmol) in DMF (15 mL) was deoxygenated byalternately freezing the mixture in a liquid nitrogen bath under vacuumand thawing under argon (3×). Bis(triphenylphosphine)palladiumdichloride (80 mg, 0.11 mmol) was added and the mixture was heated to90° C. for 4 hr as the mixture turned dark. An additional 60 mg of thecatalyst was added and heating continued for 2 hr. The mixture wasallowed to cool, was diluted with dichloromethane, and was filteredthrough a pad of celite. The filtrate was evaporated to dryness and theresidue was chromatographed over 150 g of silica gel eluting with 1:2ethyl acetate:hexane to give4-(1-benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalaninemethyl ester (255 mg, 52%). LMS-Electrospray: m/z 1015 (2M+Na) (4 Cl)519 (M+Na) 514 (M+NH4) 497 (M+H).

Example 13 4-(1-Benzyl-5-chloro-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester hydrochloride

A solution of1-benzyl-4-(5-chloro-2-oxo-3-pyridinyl)-N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalaninemethyl ester (248 mg, 0.50 mmol) in 4 N HCl in dioxane (10 mL) wasstirred for 1 hr and was concentrated. The residue was triturated withether (15 mL) and was filtered to give4-(1-benzyl-5-chloro-2-oxo-3-pyridinyl)-L-phenylalanine methyl ester(202 mg, 94%) as a white powder. LR-Electrospray: m/z 1015 (2M+Na, 4Cl), 519 (M+Na) 514 (M+NH4), 497 (M+H). HRMS: Obs. Mass 496.1774. Calcd.Mass 496.1765 (M+H).

Example 144-(1-Benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[(2-chloro-6-methylphenyl)carbonyl]-L-phenylalaninemethyl ester

A solution of 4-(1-benzyl-5-chloro-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester hydrochloride (100 mg, 0.23 mmol), 2-chloro-6-methylbenzoicacid (47 mg, 0.28 mmol), DIEA (200 μL, 1.2 mmol) and HBTU (133 mg, 0.35mmol) in DMF (2 mL) was stirred at room temperature for 18 hr. Themixture was concentrated, the residue was dissolved in ethyl acetate (15mL), was washed with 0.5 N HCl (1×5 mL), sat. NaHCO₃ (1×5 mL), brine(1×5 mL) and was dried (MgSO₄). The residue obtained upon evaporationwas chromatographed on 25 g of silica gel, eluting with 45:55 ethylacetate:hexane to give4-(1-benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[(2-chloro-6-methylphenyl)carbonyl]-L-phenylalaninemethyl ester (74 mg, 58%). Obs. Mass 397.1313. Calcd. Mass 397.1320(M+H)

Example 154-(1-Benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[(2-chloro-6-methylphenyl)carbonyl]-L-phenylalanine

A solution ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1-benzyl-5-chloro-2-oxo-pyridinyl)-L-phenylalaninemethyl ester (72 mg, 0.13 mmol) in THF (3 mL) was treated with asolution of lithium hydroxide monohydrate (22 mg, 0.52 mmol) in water(1.0 mL). Additional THF was added to effect a clear solution and thereaction mixture was stirred 1.5 hr at which time, TLC (ethyl acetate)indicated starting material was consumed. A few drops of acetic wereadded and the entire reaction mixture was applied to a 4×30 cm, C-18reversed phase HPLC column and eluted with a linear gradient ofacetonitrile in water of 5 to 95% over 35 min. The product containingfraction was lyophillyzed to give4-(1-benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[(2-chloro-6-methylphenyl)carbonyl]-L-phenylalanine(50 mg, 71%) as a white solid. LR-Electrospray: m/z negative ion 533(M−H (2 Cl)); positive ion 557(M+Na), 552 (M+NH4), 535 (M+H (2 Cl)).HRMS: Obs. Mass 535.1190. Calcd. Mass 535.1191 (M+H).

Example 164-(1-Benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalaninemethyl ester

A solution of 4-(1-benzyl-5-chloro-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester hydrochloride (100 mg, 0.25 mmol),1-(2-methoxyethyl)cyclopentane carboxylic acid (47 mg, 0.28 mmol), DIEA(200 μL, 1.2 mmol) and HBTU (133 mg, 0.35 mmol) in DMF (2 mL) wasstirred at room temperature for 18 hr. The mixture was concentrated, theresidue was dissolved in ethyl acetate (15 mL), was washed with 0.5 NHCl (1×5 mL), sat. NaHCO₃ (1×5 mL), brine (1×5 mL) and was dried(MgSO₄). The residue obtained upon evaporation was chromatographed on 25g of silica gel, eluting with 60:40 ethyl acetate:hexane to give4-(1-benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalaninemethyl ester (80 mg, 63%). LR-Electrospray: m/z 1123 (2M+Na), 1118(2M+NH4), 573 (M+Na), 568 (M+NH4), 551 (M+H (1 Cl)). HRMS: Obs. Mass551.2297. Calcd. Mass 551.2313 (M+H).

Example 174-(1-Benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine

A solution of4-(1-benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalaninemethyl ester (78 mg, 0.142 mmol) in THF (3 mL) was treated with asolution of lithium hydroxide monohydrate (24 mg, 0.57 mmol) in water(1.0 mL). Sufficient THF was added to the mixture to effect solution.The mixture was stirred 1 hr and a few drops of acetic acid were added.The entire reaction mixture was applied to a 4×30 cm, C-18 reversedphase HPLC column and eluted with a gradient of acetonitrile in water of5- to 95% over 35 min. The product containing fraction was lyophillyzedto give4-(5-chloro-1-benzyl-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine(45 mg, 60%) as a white solid. LRMS-Electrospray: m/z positive ion 559(M+Na), 554 (M+NH4), 537 (M+H (1 Cl)); negative ion 535 (M−H (1 Cl)).HRMS: obs. mass 537.2151. calcd. mass 537.2156 (M+H).

Example 18 3-Bromo-1H-2-pyridone

To a solution of 1H-2-pyridone (1.9 g, 20 mmol) in 1M aqueous potassiumbromide (20 mL) was added a solution of bromine (3.2 g, 20 mmol) in 1 Maqueous potassium bromide (40 mL) over 5 min. The reaction was allowedto proceed overnight and the resulting precipitate was collected to give1.4 g. Recrystallization from acetonitrile afforded 0.78 g of3-bromo-1H-2-pyridone.

Example 19

3-Bromo-1-methyl-2-pyridone

A mixture of 3-bromo-1H-2-pyridone (740 mg, 4.25 mmol), potassiumcarbonate (1.18 g, 8.5 mmol) and iodomethane (2.65 mL, 42.5 mmol) in DME(10 mL) was heated to reflux overnight. The mixture was filtered,washing with ethyl acetate and the filtrate was evaporated to dryness.The residue was purified by dry column chromatography over 27 g ofsilica gel, eluting with ethyl acetate to give3-bromo-1-methyl-2-pyridone (0.63 g, 79%). LRMS (electrospray), positiveion, 188 (M+H).

Example 20 3-bromo-1-methyl-2-pyridone and3,5-dibromo-1-methyl-2-pyridone

A solution of 1-methyl-2-pyridone (2.73 g, 25 mmol) and bromine (1.4 mL,27 mmol) in glacial acetic acid (150 mL) was stirred 48 hr at roomtemperature. The mixture was concentrated. The residue was taken up inwater (100 mL), made basic to litmus paper with 10 N sodium hydroxideand was extracted with dichloromethane (3×50 mL). The combined extractswere washed with brine (30 mL) and dried (MgSO₄). Filtration andevaporation of the solvent gave 4.7 g, which was purified by drychromatography over 150 g of silica gel, eluting with 1:6 ethylacetate:hexane followed by 1:1 ethyl acetate:hexane. The first compoundto elute was 3,5-dibromo-1-methyl-2-pyridone (2.0 g, 30%),LR-Electrospray: m/z 266 (M+H (2 Br)). NMR(CDCl₃) δ 3.597 (s, 3H), 7.426(d, j=2.7, 11H), 7.791 (d, j=2.7, 1H). The second compound to elute was3-bromo-1-methyl-2-pyridone (1.39 g, 30%), LR-Electrospray: m/z positiveion 251 (M+Na⁺ CH₃CN), 188 (M+H).

Example 21N-[(1,1-Dimethylethoxyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester

A solution of 3-bromo-1-methyl-2-pyridone (94 mg, 0.50 mmol) andN-[(1,1-dimethylethoxy)carbonyl]-4-[(tributyl)stannyl]-L-phenylalaninemethyl ester (284 mg, 0.50 mmol) in DMF (3 mL) was deoxygenated byalternately freezing the mixture in a liquid nitrogen bath under vacuumand thawing under argon (3×). bis(triphenylphosphine)palladiumdichloride (40 mg, 0.057 mmol) was added and the mixture was heated to90° C. for 3 hr as the mixture turned dark. TLC indicated that thereaction was not complete and an additional 20 mg portion of thecatalyst was added and heating continued for 4 hr. The mixture wasallowed to cool and was diluted with dichloromethane (20 mL) and wasfiltered through a pad of celite. The filtrate was evaporated to drynessand the residue was dissolved in 1:1 ether:ethyl acetate (20 mL). Thesolution was washed with water (1×10 mL), 5% potassium fluoride (2×5 mL)and water (1×10 mL) and was dried (MgSO₄). The residue obtained uponconcentration was chromatographed over 25 g of silica gel eluting with4:6 ethyl acetate:hexane to giveN-[(1,1-dimethylethoxyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (64 mg, 33%). LRMS-Electrospray: m/z positive ion, 795(2M+Na) 409 (M+Na) 387 (M+H). HRMS: Obs. Mass 387.1935. Calcd. Mass387.1920 (M+H).

Example 22 4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine methyl esterhydrochloride

A solution ofN-[(1,1-dimethylethoxyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (60 mg, 0.155 mmol) in 4 N HCl in dioxane (4 mL) wasstirred for 2 hr and was concentrated. The residue was triturated withseveral portions of ether to give4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine methyl esterhydrochloride (55 mg, quant) as a white powder. LRMS-Electrospray: m/zpositive ion 573 (2M+H) 328 (M+H+CH3CN) 319 (M+H+CH3OH) 287 (M+H). HRMS:Obs. Mass 287.1396. Calcd. Mass 287.1396 (M+H)

Example 23N-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester

A solution of 4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine methylester hydrochloride (52 mg, 0.16 mmol), 2-bromo-5-methoxybenzoic acid(45 mg, 0.19 mmol), DIEA (140 μL, 0.80 mmol) and HBTU (85 mg, 0.22 mmol)in DMF (3 mL) was stirred at room temperature for 18 hr. The mixture wasconcentrated, the residue was dissolved in ethyl acetate (15 mL), waswashed with 0.5 N HCl (1×5 mL), sat. NaHCO₃ (1×5 mL), brine (1×5 mL) andwas dried (MgSO₄). The residue obtained upon evaporation (85 mg) waschromatographed on 25 g of silica gel, eluting with ethyl acetate togiveN-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (54 mg, 68%). HRMS: Obs. Mass 499.0872. Calcd. Mass499.0869 (M+H)

Example 24N-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine

A solution ofN-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (52 mg, 0.104 mmol) in THF (3 mL) was treated with asolution of lithium hydroxide monohydrate (20 mg, 0.47 mmol) in water(1.0 mL). Methanol (0.5 mL) was added to effect a clear solution and thereaction mixture was stirred 18 hr. Acetic acid (0.5 mL) was added, theentire reaction mixture was applied to a 4×30 cm, C-18 reversed phaseHPLC column and eluted with a gradient of acetonitrile in water of 5 to95% over 35 min. The product containing fraction was lyophillyzed togiveN-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine(39 mg, 78%) as a white solid. HRMS: Obs. Mass 485.0703 Calcd. Mass485.0712 (M+H).

Example 254-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-N-[(1,1-dimethylethoxyl)carbonyl]-L-phenylalaninemethyl ester

solution of 3,5-dibromo-1-methyl-2-pyridone (267 mg, 1.0 mmol) andN-[(1,1-dimethylethoxy)carbonyl]-4-[(tributyl)stannyl]-L-phenylalaninemethyl ester (568 mg, 1.0 mmol) in DMF (7 mL) was deoxygenated byalternately freezing the mixture in a liquid nitrogen bath under vacuumand thawing under argon (3×). bis(triphenylphosphine)palladiumdichloride (80 mg, 0.11 mmol) was added and the mixture was heated to90° C. for 3 hr as the mixture turned dark. TLC indicated that thereaction was not complete and an additional 40 mg portion of thecatalyst was added and heating continued for 4 hr. The mixture wasallowed to cool and was diluted with dichloromethane (40 mL) and wasfiltered through a pad of celite. The filtrate was evaporated to drynessand the residue was dissolved in ethyl acetate (30 mL). The solution waswashed with water (1×10 mL), 5% potassium fluoride (2×10 mL) and brine(1×5 mL) and was dried (MgSO₄). The residue obtained upon concentrationwas chromatographed over 45 g of silica gel eluting with 3:7 ethylacetate:hexane to give4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-N-[(1,1-dimethylethoxyl)carbonyl]-L-phenylalaninemethyl ester (140 mg, 30%). FAB MS: 465 (M+H (1 Br)).

Example 26 4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine methylester hydrochloride

A solution ofN-[(1,1-dimethylethoxyl)carbonyl]-4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (135 mg, 0.29 mmol) in 4 N HCl in dioxane (5 mL) wasstirred for 2 hr and was concentrated. The residue was triturated withseveral portions of ether to give4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine methyl esterhydrochloride (120 mg, quant) as a white powder. LRMS-Electrospray: m/zpositive ion 729, 2M+H (2 Br), 406 (M+H+CH3CN), 397 (M+H+CH3CN (1 Br)),365 (M+H (1 Br)). HRMS: Obs. Mass 365.0504. Calcd. Mass 365.0501 (M+H).

Example 27N-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester

A solution of 4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester hydrochloride (120 mg, 0.30 mmol), 2-bromo-5-methoxybenzoicacid (82 mg, 0.36 mmol), DIEA (260 μL, 1.5 mmol) and HBTU (157 mg, 0.41mmol) in DMF (4 mL) was stirred at room temperature for 18 hr. Themixture was concentrated, the residue was dissolved in ethyl acetate (15mL), was washed with 0.5 N HCl (1×5 mL), sat. NaHCO₃ (1×5 mL), brine(1×5 mL) and was dried (MgSO₄). The residue obtained upon evaporation(180 mg) was chromatographed on 25 g of silica gel, eluting with 3:1ethyl acetate:hexane to giveN-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (130 mg, 76%). HRMS: Obs. Mass 576.9959. Calcd. Mass576.9973 (M+H)

Example 28N-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine

A solution ofN-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (29 mg, 0.05 mmol) in THF (3 mL) was treated with asolution of lithium hydroxide monohydrate (20 mg, 0.47 mmol) in water(1.0 mL). Methanol (0.5 mL) was added to effect a clear solution and thereaction mixture was stirred 18 hr. Acetic acid (0.5 mL) was added, theentire reaction mixture was applied to a 4×30 cm, C-18 reversed phaseHPLC column and eluted with a gradient of acetonitrile in water of 5 to95% over 35 min. The product containing fraction was lyophillyzed togiveN-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine(22 mg, 79%) as a white solid. HRMS: Obs. Mass 562.9814. Calcd. Mass562.9817 (M+H).

Example 29 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester

To a suspension of 4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine methylester hydrochloride salt (101 mg, 0.313 mmol), 2-chloro-6-methylbenzoicacid (60 mg, 0.35 mmol) and HBTU (133 mg, 0.35 mmol) in DMF (2 mL) wasadded DIEA (122 μL, 0.88 mmol) at room temperature. The resultingmixture was stirred for 15 h. Then, the mixture was poured into water(25 mL) and the organic compound was extracted into ethyl acetate (2×15mL). The combined ethyl acetate extracts were washed successively with0.5 N HCl (25 mL), saturated NaHCO₃ solution (25 mL), brine solution (25mL) and were dried over anhydrous magnesium sulfate. Filtration of thedrying agent and concentration gave the crude product which was purifiedby silica gel chromatography using a Biotage (40 s) column to afford 122mg (88% yield) ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester as an amorphous white solid. FAB-HRMS m/e calcd forC₂₄H₂₃ClN₂O₄ (M+H) 439.1425. found 439.1414.

Example 30 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine

To a suspension ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (118 mg, 0.269 mmol) in ethanol (6 mL) was added 1N aqueoussodium hydroxide solution (4 mL) at room temperature. The resultingsolution was heated to 50° C. and stirred for 2 h. Then, the ethanol wasremoved under vacuum and the residue was diluted with water (25 mL). Theaqueous solution was washed with diethyl ether (25 mL) to remove anyneutral impurities. The aqueous layer was acidified with 1.0 N HCl andthe product was extracted into ethyl acetate (2×25 mL). The combinedorganic extracts were washed with brine solution (50 mL) and were driedover anhydrous magnesium sulfate. Filtration of the drying agent andconcentration afforded 81 mg (71% yield) ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanineas a white solid: mp 204-210° C. FAB-HRMS m/e calcd for C₂₃H₂₁ClN₂O₄(M+H) 425.1268. found 425.1267.

Example 31 Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalanine

Preparation of 1,4-dimethyl-2(1H)-pyridone

To a suspension of 4-methy-2(1H)-pyridone (5 g, 45.82 mmol) andpotassium carbonate (12.64 g, 91.64 mmol) in DME (100 mL) was addediodomethane (50.5 g, 366 mmol) at room temperature and the reactionmixture was heated to reflux overnight. The reaction mixture was cooledto room temperature, poured into water (200 mL) and was extracted withethyl acetate (3×100 mL). The combined extracts were washed with brinesolution (200 mL) and were dried over anhydrous magnesium sulfate.Filtration of the drying agent and concentration gave the crude productwhich was purified by silica gel chromatography on a Biotage (40 m)column to afford 2.5 g (44% yield) of 1,4-dimethyl-2(1H)-pyridone as anamorphous white solid. FAB-HRMS m/e calcd for C₇H₉NO (M+H) 123.0241.found 123.0246.b) Preparation of 1,4-dimethyl-3-iodo-2(1H)-pyridone and1,4-dimethyl-5-iodo-2(1H)-pyridone

A reaction mixture containing 1,4-dimethyl-2-pyridone (2.46 g, 20 mmol),trifluoroacetic acid (31 mL) and trifluoroacetic anhydride (6.25 mL) wasrefluxed for 5 min. Then, NIS (5.62 g, 25 mmol) was added and themixture was stirred for 15 h. The reaction mixture was cooled to roomtemperature and the solvent was removed under vacuum. The residue wasdiluted with ethyl acetate (100 mL) and the white solid that formed wascollected by filtration. The filtrate was washed with saturated sodiumbicarbonate solution (2×100 mL), brine solution (100 mL) and was driedover anhydrous magnesium sulfate. Filtration of the drying agent andconcentration of the solvent gave a crude product which was purified bysilica gel chromatography on a Biotage (40 m) column to afford 0.92 g(5% yield) of a ˜1:1 mixture of 1,4-dimethyl-3-iodo-2(1H)-pyridone and1,4-dimethyl-5-iodo-2(1H)-pyridone which was used directly in the nextstep. ¹H NMR (300 MHz, D6-DMSO, ppm) 8.06 (s, 1H), 7.6 (d, 1H, J=5.5Hz), 6.4 (s, 1H), 6.2 (d, 1H, J=5.5 Hz), 3.4 (s, 3H), 3.3 (s, 3H), 2.3(s, 3H), 2.15 (s, 3H).c) Preparation ofN-[(1,1-dimethylethoxyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester andN-[(1,1-dimethylethoxyl)carbonyl]-4-(1,4-dimethyl-2-oxo-5-pyridinyl)-L-phenylalaninemethyl ester

To a suspension of zinc dust (0.66 g, 10 mmol) in THF (1.0 mL) was added1,2-dibromoethane (86 μL, 1 mmol)) at room temperature. This suspensionwas heated to 60-65° C. with a heat gun until evolution of ethylene gasceased. Then, the suspension was cooled to room temperature andtrimethylchlorosilane (0.126 mL, 1 mmol)) was added and the mixture wasstirred for 15 min. A mixture of 1,4-dimethyl-3-iodo-2(1H)-pyridone and1,4-dimethyl-5-iodo-2(1H)-pyridone (0.92 g, 3.69 mmol) in DMA (3 mL) waswarmed to effect dissolution and was added in one portion to thereaction mixture. After addition, the mixture was heated to 70° C. andwas stirred for 15 h, at which time the TLC analysis of an aliquot,which had been quenched with saturated ammonium chloride solution,indicated the absence of starting material. The reaction mixture wasdiluted with THF (4 mL) and was cooled to room temperature. The excesszinc dust was allowed to settle until a clear supernatant liquid formed(˜3 h).

The above prepared solution containing the zinc compound (3.69 mmol) wasadded to a solution of Pd(dba)₂ (54 mg, 0.1 mmol), trifurylphosphine(102 mg, 0.4 mmol) andN-[(1,1-dimethylethoxy)carbonyl]-4-iodo-L-phenylalanine methyl ester(1.01 g, 2.5 mmol) in THF (4 mL) at room temperature and the lightyellow mixture was stirred for 15 h at 50° C. Then, the mixture waspoured into a saturated ammonium chloride solution and was extractedwith ethyl acetate (3×50 mL). The combined extracts were washed withbrine solution (150 mL) and were dried over anhydrous magnesium sulfate.Filtration of the drying agent and concentration gave the crude productwhich was purified by silica gel chromatography on a Biotage (40 m)column to obtain 0.141 g (14% yield) ofN-[(1,1-dimethylethoxyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester as an amorphous white solid. ES-HRMS m/e calcd forC₂₂H₂₈N₂O₅ (M+Na) 423.1890. found 423.1894 and 0.350 g (35% yield) ofN-[(1,1-dimethylethoxyl)carbonyl]-4-(1,4-dimethyl-2-oxo-5-pyridinyl)-L-phenylalaninemethyl ester as an amorphous white solid. ES-HRMS m/e calcd forC₂₂H₂₈N₂O₅ (M+Na) 423.1890. found 423.1897.d) Preparation of 4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester hydrochloride salt

To a solution ofN-[(1,1-dimethylethoxyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (132 mg, 0.33 mmol) in dioxane (1 mL) was added 4 N HClsolution in dioxane (1.5 mL) at room temperature. The solution wasstirred for 4 h and concentrated under vacuum. The residue was dissolvedin methanol (5 ml) and toluene (5 mL) and concentrated under vacuum togive 111 mg (99% yield) of4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalanine methyl esterhydrochloride salt as an amorphous white solid. EI-HRMS m/e calcd forC₁₇H₂₀N₂O₃ (M⁺) 300.1474. found 300.1486.e) Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester

To a suspension of 4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester hydrochloride salt (34 mg, 0.1 mmol) and2,6-dichlorobenzoyl chloride (25 mg, 0.12 mmol) in dichloromethane (2mL) was added DIEA (174 μL, 1.0 mmol) at room temperature. After 5 min,a clear solution was obtained which was stirred for 72 h. Then, themixture was concentrated, the residue was dissolved in ethyl acetate (25mL), was washed with 0.5 N HCl (25 mL), saturated NaHCO₃ solution (25mL), brine solution (15 mL) and was dried over anhydrous magnesiumsulfate. Filtration of the drying agent and concentration gave crudeproduct, which was purified by silica gel chromatography using a Biotage(40 s) column, to afford 23 mg (49% yield) ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester as an amorphous white solid. ES-HRMS m/e calcd forC₂₄H₂₂Cl₂N₂O₄ (M+Na) 495.0850. found 495.0859.f) Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalanine

To a suspension ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (20 mg, 0.04 mmol) in ethanol (1 mL) was added 1N aqueoussodium hydroxide solution (0.5 mL) at room temperature. The mixture washeated to 40-45° C. and stirred for 3 h. Then, the ethanol was removedunder vacuum and the residue was diluted with water (10 mL). The aqueoussolution was washed with diethyl ether (25 mL) to remove any neutralimpurities. The aqueous layer was acidified with 1.0 N HCl and theproduct was extracted into ethyl acetate (2×25 mL). The combined organicextracts were washed with brine solution (50 mL) and were dried overanhydrous magnesium sulfate. Filtration of the drying agent andconcentration afforded 17 mg (89% yield) ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalanineas an amorphous white solid. ES-HRMS m/e calcd for C₂₃H₂₀Cl₂N₂O₄ (M+Na)481.0691. found 481.0699.

Example 32 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalanine

Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester

To a suspension of 4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester hydrochloride salt (68 mg, 0.2 mmol),2-chloro-6-methylbenzoic acid (45 mg, 0.25 mmol) and HBTU (95 mg, 0.25mmol) in DMF (1.5 mL) was added DIEA (174 μL, 1.0 mmol) at roomtemperature. The resulting solution was stirred for 72 h, the mixturewas poured into water (25 mL) and was extracted with ethyl acetate (2×15mL). The combined ethyl acetate extracts were washed successively with0.5 N HCl (25 mL), saturated NaHCO₃ solution (25 mL), brine solution (25mL) and were dried over anhydrous magnesium sulfate. Filtration of thedrying agent and concentration of the solvent gave the crude product,which was purified by silica gel chromatography using a Biotage (40 s)column to afford 24 mg (27% yield) ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester as an amorphous white solid. ES-HRMS m/e calcd forC₂₅H₂₅ClN₂O₄ (M+Na) 475.1395. found 475.1400.b) Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalanine

To a suspension ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalaninemethyl ester (22 mg, 0.048 mmol) in ethanol (1 mL) was added 1N aqueoussodium hydroxide solution (0.5 mL) at room temperature. The resultingsolution was heated to 40-45° C. and stirred for 2 h. Then, the ethanolwas removed under vacuum and the residue was diluted with water (25 mL).The aqueous solution was washed with diethyl ether (25 mL) to remove anyneutral impurities. The aqueous layer was acidified with 1.0 N HCl andthe product was extracted into ethyl acetate (2×25 mL). The combinedextracts were washed with brine solution (50 mL) and were dried overanhydrous magnesium sulfate. Filtration of the drying agent andconcentration afforded 18 mg (86% yield) ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalanineas an amorphous white solid. ES-HRMS m/e calcd for C₂₄H₂₃ClN₂O₄ (M+H)439.1419. found 439.1425.

Example 33 Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

Preparation of 1,6-dimethyl-4-(trifluoromethyl)-2-pyridone

To a suspension of 6-methyl-4-(trifluoromethyl)-1H-2-pyridone (2 g,11.25 mmol) and potassium carbonate (4.68 g, 33.9 mmol) in DME (25 mL)was added iodomethane (9.62 g, 67.8 mmol) at room temperature and thereaction mixture was heated to reflux overnight. The reaction mixturewas cooled to room temperature, poured into water (100 mL) and extractedwith ethyl acetate (3×100 mL). The combined extracts were washed withbrine solution (200 mL) and were dried over anhydrous magnesium sulfate.Filtration of the drying agent and concentration of the solvent gave thecrude product which was purified by silica gel chromatography on aBiotage (40 m) column to obtain 2.1 g (97% yield) of1,6-dimethyl-4-(trifluoromethyl)-2-pyridone as a white solid: mp 80-82°C. EI-HRMS m/e calcd for C₈H₈F₃NO (M⁺) 191.0558. found 191.0559.b) Preparation of 1,6-dimethyl-3-iodo-4-(trifluoromethyl)-2-pyridone

A mixture containing 1,6-dimethyl-4-(trifluoromethyl)-2-pyridone (2.1 g,10.98 mmol), trifluoroacetic acid (18 mL) and trifluoroacetic anhydride(3.5 mL) was refluxed for 5 min. Then, NIS (3.15 g, 14 mmol) was addedand the reaction mixture was stirred for 15 h. The reaction mixture wascooled to room temperature and the solvent was removed under vacuum. Theresidue was diluted with ethyl acetate (100 mL), was washed withsaturated sodium bicarbonate solution (2×100 mL) and brine solution (100mL) and was dried over anhydrous magnesium sulfate. Filtration of thedrying agent and concentration gave a crude product, which was purifiedby silica gel chromatography on a Biotage (40 m) column to afford 2.15 g(62% yield) of 1,6-dimethyl-3-iodo-4-(trifluoromethyl)-2-pyridone as anamorphous white solid. EL-HRMS m/e calcd for C₈H₇F₃INO (M⁺) 316.9524.found 316.9527.c) Preparation ofN-[(1,1-dimethylethoxyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester

To a suspension of zinc dust (1.96 g, 30 mmol) in THF (2.0 mL) was added1,2-dibromoethane (172 μL, 2 mmol)) at room temperature. This suspensionwas heated to 60-65° C. with a heat gun until evolution of ethylene gasceased. Then, the suspension was cooled to room temperature andtrimethylchlorosilane (127 μL, 1 mmol)) was added and the mixture wasstirred for 15 min. A suspension of1,6-dimethyl-3-iodo-4-(trifluoromethyl)-2-pyridone (2.15 g, 6.78 mmol)in DMA (6 mL) was warmed with a heat gun to effect dissolution and wasadded in one portion to the reaction mixture. After addition, themixture was heated to 70° C. and was stirred for 15 h, at which time theTLC analysis of an aliquot, which had been quenched with saturatedammonium chloride solution, indicated the absence of starting material.The reaction mixture was diluted with THF (6 mL), was cooled to roomtemperature and the excess zinc dust was allowed to settle.

The above prepared solution containing the zinc compound (6.78 mmol) wasadded to a solution of Pd(dba)₂ (108 mg, 0.2 mmol), trifurylphosphine(204 mg, 0.8 mmol) andN-[(1,1-dimethylethoxy)carbonyl]-4-iodo-L-phenylalanine methyl ester(1.62 g, 4 mmol) in THF (8 mL) at room temperature and the light yellowmixture was stirred for 15 h at 50° C. The reaction mixture was pouredinto a saturated ammonium chloride solution and was extracted with ethylacetate (3×70 mL). The combined extracts were washed with brine solution(150 mL) and were dried over anhydrous magnesium sulfate. Filtration ofthe drying agent and concentration gave the crude product, which waspurified by silica gel chromatography on a Biotage (40 m) column toobtain 0.711 g (38% yield) ofN-[(1,1-dimethylethoxyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester as an amorphous white solid. ES-HRMS m/e calcd forC₂₃H₂₇F₃N₂O₅ (M+Na) 491.1764. found 491.1770.d) Preparation of4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

methyl ester hydrochloride salt

To a solution ofN-[(1,1-dimethylethoxyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester (132 mg, 0.33 mmol) in dioxane (3 mL) was added 4 N HClsolution in dioxane (4.5 mL) at room temperature. The solution wasstirred for 4 h as a white solid was formed. The mixture was dilutedwith diethyl ether (50 mL) and solid was collected by filtration washingwith diethyl ether. After drying under high vacuum, 315 mg (92% yield)of4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester hydrochloride salt was obtained as an amorphous whitesolid. ES-HRMS m/e calcd for C₁₈H₁₉F₃N₂O₃ (M+Na) 391.1241. found391.1241.e) Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester

To a suspension of4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester hydrochloride salt (150 mg, 0.37 mmol) and2,6-dichlorobenzoyl chloride (85 mg, 0.4 mmol) in dichloromethane (6 mL)was added DIEA (257 μL, 1.48 mmol) at room temperature. After 5 min, aclear solution was obtained which was stirred for 48 h. Then, themixture was concentrated and the residue was dissolved in ethyl acetate(50 mL). The ethyl acetate solution was washed with 0.5 N HCl (50 mL),saturated NaHCO₃ solution (50 mL) and brine solution (50 mL) and wasdried over anhydrous magnesium sulfate. Filtration of the drying agentand concentration gave crude product, which was purified by silica gelchromatography on a Biotage (40 m) column to give 190 mg (95% yield) ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester as an amorphous white solid. ES-HRMS m/e calcd forC₂₅H₂₁Cl₂F₃N₂O₄ (M+Na) 563.0724. found 563.0726.f) Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

To a suspension ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester (177 mg, 0.326 mmol) in ethanol (6 mL) was added 1N aqueoussodium hydroxide solution (4 mL) at room temperature. The mixture wasstirred for 5 h. Then, the ethanol was removed under vacuum and theresidue was diluted with water (20 mL). The aqueous solution was washedwith diethyl ether (50 mL) to remove any neutral impurities. The aqueouslayer was acidified with 1.0 N HCl and the product was extracted withethyl acetate (2×50 mL). The combined organic extracts were washed withbrine solution (50 mL) and were dried over anhydrous magnesium sulfate.Filtration of the drying agent and concentration afforded 159 mg (92%yield) ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineas a white solid: mp 238-240° C. ES-HRMS m/e calcd for C₂₄H₁₉Cl₂F₃N₂O₄(M+Na) 549.0567. found 549.0570.

Example 34 Preparation ofN-[(2-ethyl-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

a. Preparation of 2-ethyl-6-methylbenzoic acid.

A 250 mL pressure bottle was charged with 2-ethyl-6-methyliodobenzene(30.07 mmol, 7.4 g), Pd(OAc)₂ (1.43 mmol, 334 mg) and dppp (1.43 mmol,620 mg). The flask was closed with a septum and evacuated three timeswith argon. Then, acetonitrile (96 mL), triethylamine (189 mmol, 19.0 g,26.25 mL) and water (19.1 mL) were added successively by the aid ofsyringe and the rubber septum was replaced with teflon lined capconnected to a carbon monoxide source. The flask was now pressurizedwith carbon monoxide (40 psi) and the excess pressure was released. Thisprocess was repeated three times and finally the mixture was stirred for5 min under 40 psi carbon monoxide pressure. The flask was thendisconnected from the carbon monoxide cylinder and immersed in apreheated oil bath (83-85° C.). The reaction mixture turned black over 1hr and was stirred for another 14 hr at this temperature. Then, thereaction mixture was cooled to room temperature and the pressure wasreleased. The resulting mixture was diluted with ether (200 mL) and 1.0NNaOH (20 mL). The formed acid was extracted into water (2×100 mL). Thecombined water extracts were neutralized with 1.0N HCl and the acid wasextracted into dichloromethane (3×100 mL). The combined dichloromethaneextracts were washed with brine solution and dried over MgSO₄.Filtration of the drying agent and removal of solvent under vacuum gave3.58 g (72.5%) of a viscous brown oil which slowly solidfied overnight.HR MS: Obs. mass, 164.0833. Calcd. mass, 164.0837 (M+).b. Preparation of 2-ethyl-6-methylbenzoyl chloride

A solution of 2-ethyl-6-methylbenzoic acid (49 mg, 0.30 mmol) indichloromethane (3 mL) containing DMF (1 drop) was treated with oxalylchloride (0.14 mL, 1.6 mmol) and the mixture was stirred for 15 h. Themixture was concentrated, azeotroping with toluene to remove traces ofoxalyl chloride and the residue was used directly in the next step.c. Preparation ofN-[(2-ethyl-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester

A mixture of the above prepared acid chloride,4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-phenylalaninemethyl ester hydrochloride (100 mg, 0.25 mmol) in dichloromethane (5 mL)was treated with DIPEA (0.17 mL, 1.0 mmol) and the resulting light brownsolution was stirred for 3 days. The mixture was concentrated, dilutedwith ethyl acetate, washed with 1 N HCl and brine solution and was driedover magnesium sulfate. Filtration and evaporation afforded a residue,which was purified by silica gel chromatography using a Biotage column(40 s) to giveN-[(2-ethyl-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester as a white foam (79 mg, 62%). ES-HRMS m/e calcd forC₂₈H₂₉F₃NO₄ (M+Na) 537.1974. found 537.1972.d. Preparation ofN-[(2-ethyl-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

A solution ofN-[(2-ethyl-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester (74 mg, 0.14 mmol), and 1 N sodium hydroxide (2 mL, 2 mmol)in ethanol (3 mL) was heated to 40-45 C for 3 h. Then, the ethanol wasremoved under vacuum and the residue was diluted with water. The aqueoussolution was washed with diethyl ether to remove any neutral impurities.The aqueous layer was acidified with 1.0 N HCl and the product wasextracted with ethyl acetate. The combined organic extracts were washedwith brine solution and were dried over anhydrous magnesium sulfate.Filtration of the drying agent and concentration afforded 68 mg (95%yield) ofN-[(2-ethyl-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine(mp 219-221° C.). ES-HRMS m/e calcd for C₂₇H₂₇F₃N₂O₄ (M+Na) 523.1815.found 523.1816.

Example 35 Preparation ofN-[(2-(1-methylethyl)-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

a. Preparation of 2-(1-methylethyl)-6-methyliodobenzene

To a suspension of 2-(1-methylethyl)-6-methylaniline (15.57 mmol, 14.9g), in conc. HCl (50 mL) and 30 g of ice, was added dropwise a solutionof NaNO₂ (110 mmol, 8 g) in H₂O (35 mL) at −5° C. to 5° C. for 30 min.After addition, the red colored solution was stirred for another 30 min.Then, a solution of KI (200 mmol, 33.2 g) in H₂O (50 mL) was addeddropwise over 20 min at 0-5° C. After the addition, the mixture wasallowed to warm to room temperature during which time, an exothermicreaction with gas evolution occurred. The resulting red colored solutionwas stirred for 18 h. Then, the mixture was extracted with ethyl acetate(3×100 mL). The combined extracts were washed with sodium thiosulfatesolution (200 mL), brine solution and dried over MgSO₄. Filtration ofthe drying agent and concentration of the solvent under vacuum gave acolored compound which was purified by a silica gel columnchromatography to obtain pure 2-(1-methylethyl)-6-methyliodobenzene(17.8 g, 68%) as a yellow oil. HR MS: Obs. mass, 260.0063. Calcd. mass,260.0062 (M+).b. Preparation of 2-(1-methylethyl)-6-methylbenzoic acid.

A 250 mL pressure bottle was charged with2-(1-methylethyl)-6-methyliodobenzene (25.2 mmol, 6.55 g), Pd(OAc)₂ (1.2mmol, 280 mg) and dppp (1.2 mmol, 520 mg). The flask was closed with aseptum and evacuated three times with argon. Then, acetonitrile (96 mL),triethylamine (188.7 mmol, 19.0 g, 26.25 mL) and water (19.1 mL) wereadded successively by the aid of syringe. Then, the rubber septum wasreplaced with teflon lined cap connected to a carbon monoxide source.The flask was now pressurized with carbon monoxide (40 psi) and theexcess pressure was released. This process was repeated three times andfinally the mixture was stirred for 5 min under 40 psi carbon monoxidepressure. The flask was then disconnected from the carbon monoxidecylinder and immersed in a preheated oil bath (83-85° C.). The reactionmixture turned black in 1 hr and was stirred for another 4 hr at thistemperature. Then, the reaction mixture was cooled to room temperature,the pressure was released and the mixture was diluted with ether (200mL) and 1.0N NaOH (10 mL). The acid was extracted into water (2×100 mL).The combined aqueous extracts were neutralized with 1.0N HCl and theacid was extracted into ethyl acetate (2×100 mL). The combined organicextracts were washed with brine solution and dried over MgSO₄.Filtration of the drying agent and concentration gave 2.8 g (62%) of aviscous yellow oil. HR MS: Obs. mass, 178.0996. Calcd. mass, 178.0994(M+).c. Preparation of 2-(1-methylethyl)-6-methylbenzoyl chloride

A solution of 2-(1-methylethyl)-6-methylbenzoic acid (64 mg, 0.35 mmol)in dichloromethane (3 mL) containing DMF (2 drops) was treated withoxalyl chloride (0.16 mL, 1.8 mmol) and the mixture was stirred for 15h. The mixture was concentrated, azeotroping with toluene to removetraces of oxalyl chloride and the residue was used directly in the nextstep.d. Preparation ofN-[[2-(1-methylethyl)-6-methylphenyl]carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

N-[[2-(1-methylethyl)-6-methylphenyl]carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninewas prepared from4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester and 2-(1-methylethyl)-6-methylbenzoyl chloride using thegeneral procedures described in example 33. ES-HRMS m/e calcd forC₂₈H₂₉F₃N₂O₄ (M+Na) 537.1972. found 537.1977.

Example 36 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester

To a suspension of4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester hydrochloride salt (100 mg, 0.25 mmol),2-chloro-6-methylbenzoic acid (60 mg, 0.35 mmol) and HBTU (132 mg, 0.35mmol) in DMF (2 mL) was added DIEA (174 μL, 1.0 mmol) at roomtemperature. The resulting mixture was stirred for 72 h. Then, themixture was poured into water (25 mL) and was extracted with ethylacetate (2×15 mL). The combined ethyl acetate extracts were washedsuccessively with 0.5 N HCl (25 mL), saturated NaHCO₃ solution (25 mL),brine solution (25 mL) and were dried over anhydrous magnesium sulfate.Filtration of the drying agent and concentration gave the crude productwhich was purified by silica gel chromatography using a Biotage (40 s)column to afford 98 mg (75% yield) ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester as an amorphous white solid. ES-HRMS m/e calcd forC₂₆H₂₄ClF₃N₂O₄ (M+Na) 543.1268. found 543.1275.b) Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

To a suspension ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester (91 mg, 0.174 mmol) in ethanol (5 mL) was added 1N aqueoussodium hydroxide solution (4 mL) at room temperature. The resultingsolution was heated to 40-45° C. and stirred for 4 h. Then, the ethanolwas removed under vacuum and the residue was diluted with water (25 mL).The aqueous solution was washed with diethyl ether (25 mL) to remove anyneutral impurities. The aqueous layer was acidified with 1.0 N HCl andwas extracted with ethyl acetate (2×25 mL). The combined organicextracts were washed with brine solution (50 mL) and were dried overanhydrous magnesium sulfate. Filtration of the drying agent andconcentration of the filtrate afforded 71 mg (80% yield) ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineas a white solid: mp 220-223° C. ES-HRMS m/e calcd for C₂₅H₂₂ClF₃N₂O₄(M+Na) 529.1111. found 529.1119.

Example 37 Preparation ofN-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine

N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine(mp 181-183° C.) was prepared from4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester and 1-(2-methoxyethyl)cyclopentane carboxylic acid(obtained as described in WO 9910313) using the general proceduresdescribed in example 36. ES-HRMS m/e calcd for C₂₆H₃₁F₃N₂O₅ (M+Na)531.2077. found 531.2084.

Example 38 Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester

To a suspension ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine(145 mg, 0.275 mmol) and sodium bicarbonate (185 mg, 2.2 mmol) in DMF (2mL) was added iodoethane (343 mg, 2.2 mmol) at room temperature. Themixture was stirred for 72 h at room temperature. Then, the reactionmixture was poured into water (30 mL) and was extracted with ethylacetate (3×20 mL). The combined organic extracts were washed with brinesolution (60 mL) and were dried over anhydrous magnesium sulfate.Filtration of the drying agent and concentration of the filtrate gave129 mg (85% yield) ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester as a crystalline white solid: mp 86-91° C. ES-HRMS m/e calcdfor C₂₆H₂₃Cl₂F₃N₂O₄ (M+Na) 577.0876. found 577.0887.

Example 39 Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester

To a mixture ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine(145 mg, 0.275 mmol), 2-diethylaminoethyl chloride hydrochloride (487mg, 2.75 mmol) and potassium carbonate (380 mg, 2.7 mmol) was addedethyl acetate (3 mL) and water (3 mL) at room temperature. The mixturewas stirred for 72 h at room temperature. Then, the reaction mixture waspoured into a mixture of water (30 mL) and ethyl acetate (30 mL). Thelayers were separated and the aqueous layer was extracted with ethylacetate (2×20 mL). The combined organic extracts were washed with brinesolution (60 mL) and were dried over anhydrous magnesium sulfate.Filtration of the drying agent and concentration gave the crude product,which was purified by silica gel chromatography using a Biotage (40 s)column afforded 122 mg (71% yield) ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester as an amorphous white solid. ES-HRMSm/e calcd for C₃₀H₃₂Cl₂F₃N₃O₄ (M+H) 626.1796. found 626.1802.

Example 40 Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester

To a suspension ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine(145 mg, 0.275 mmol) and sodium bicarbonate (185 mg, 2.2 mmol) in DMF (2mL) was added 1-chloroethyl acetate (270 mg, 2.2 mmol) at roomtemperature. The mixture was stirred for 48 h at room temperature. Then,the reaction mixture was poured into water (30 mL) and was extractedwith ethyl acetate (3×20 mL). The combined organic extracts were washedwith brine solution (60 mL) and were dried over anhydrous magnesiumsulfate. Filtration of the drying agent and concentration gave the crudeproduct, which was purified by silica gel chromatography using a Biotage(40 s) column to afford 110 mg (65% yield) ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester as an amorphous white solid. ES-HRMS m/e calcdfor C₂₈H₂₅Cl₂F₃N₂O₆ (M+Na) 635.0931. found 635.0932.

Example 41 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester

N-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester can be prepared fromN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineand iodoethane using the general procedure described in example 38.

Example 42 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester

N-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester can be prepared fromN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineand 2-[(N,N-diethyl)amino]ethyl chloride hydrochloride using the generalprocedure described in example 39.

Example 43 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester

N-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester can be prepared fromN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineand 1-chloroethyl acetate using the general procedure described inexample 40.

Example 44 Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl)-L-phenylalanine

Preparation of 4-methoxy-1,1,1-trifluoropent-3-en-2-one

To a solution of 2-methoxypropene (3.68 g, 51.03 mmol) and pyridine(1.35 g, 16.69 mmol) in dichloromethane (15 mL) was added a solution oftrifluoroacetic anhydride (10 mL, 46.56 mmol) in dichloromethane (8 mL)at 0° C. over a period of 10-12 min. After addition, the solution turnedto dark red-brown and then the cooling bath was removed and stirring wasstopped. The mixture was allowed to stand for 16 h and was diluted withice cold water (45 mL) and dichloromethane (120 mL). The two layers wereseparated and the organic layer was washed successively with 2N HCl (35mL), saturated sodium carbonate solution (75 mL) and brine solution (25mL) and was dried over anhydrous sodium sulfate. Filtration of thedrying agent and concentration gave the crude product, which waspurified by distillation under high vacuum to afford 5.546 g (65% yield)of 4-methoxy-1,1,1-trifluoropent-3-en-2-one as a light yellow oil.

b) Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl)-L-phenylalanine

Preparation of 4-methoxy-1,1,1-trifluoropent-3-en-2-one

To a suspension of 4-methoxy-1,1,1-trifluoropent-3-en-2-one (5.53 g,32.89 mmol) and ethyl malonate monoamide (4.31 g, 32.89 mmol) in ethanol(30 mL) was added sodium ethoxide (11.72 g, 36.18 mmol, 21% pure) atroom temperature and the reaction mixture was heated to ˜85° C. Afterstirring for 18 h, the reaction mixture was cooled to room temperatureand 15% HCl (10 mL) was added. Then, it was diluted with water (10 mL)and was extracted with chloroform (2×50 mL). The combined extracts werewashed with brine solution (100 mL) and were dried over anhydrous sodiumsulfate. Filtration of the drying agent and concentration gave the crudeproduct, which was purified by silica gel chromatography on a Biotage(40 m) column to afford 5.22 g (62% yield) of4-methyl-2(1H)-oxo-6-(trifluoromethyl)pyridine-3-carboxylic acid ethylester as an amorphous white solid. ES-LRMS: m/z 313.4 (M+Na+CH₃CN).

Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl)-L-phenylalanine

Preparation of 4-methoxy-1,1,1-trifluoropent-3-en-2-one

To a suspension of4-methyl-2(1H)-oxo-6-(trifluoromethyl)pyridine-3-carboxylic acid ethylester (5.2 g, 20.87 mmol) and potassium carbonate (8.65 g, 62.59 mmol)in DME (50 mL) was added iodomethane (12 mL, 192.8 mmol) at roomtemperature and the reaction mixture was heated to reflux for 18 h. Thereaction mixture was cooled to room temperature and the inorganic solidswere filtered and the solids were washed with DME. The solvent wasconcentrated under vacuum and the residue was purified by silica gelchromatography on a Biotage (40 m) column to afford 4.02 g (71% yield)of 1,4-dimethyl-2(1H)-oxo-6-(trifluoromethyl)pyridine 3-carboxylic acidethyl ester as an amorphous white solid. ES-HRMS m/e calcd forC₁₁H₁₂F₃NO₃ (M+Na) 286.0661. found 286.0664.d) Preparation of 1,4-dimethyl-6-(trifluoromethyl)-1H-pyridin-2-one

To a mixture of1,4-dimethyl-2(1H)-oxo-6-(trifluoromethyl)pyridine-3-carboxylic acidethyl ester (2.5 g, 9.5 mmol) and lithium chloride (1.0 g, 23.6 mmol)was added DMF (15 mL) and water (0.38 mL) at room temperature. Thereaction mixture was heated to a bath temperature of 160° C. and stirredfor 19 h. The reaction mixture was cooled to room temperature anddiluted with cold ethyl acetate and diethyl ether (75 mL, 1:1). Theresulting mixture was washed with cold water (3×20 mL) and brinesolution (20 mL) and was dried over anhydrous sodium sulfate. Filtrationof the drying agent and concentration of the solvent gave the crudeproduct, which was purified by silica gel chromatography on a Biotage(40 s) column to afford 1.45 g (80% yield) of1,4-dimethyl-6-(trifluoromethyl)-1H-pyridin-2-one as a light yellowsolid. ES-HRMS m/e calcd for C₈H₈F₃NO (M+H) 192.0631. found 192.0632.e) Preparation of1,4-dimethyl-3-iodo-6-(trifluoromethyl)-1H-pyridin-2-one

To a solution of 1,4-dimethyl-6-(trifluoromethyl)-1H-pyridin-2-one (1.44g, 7.23 mmol), trifluoroacetic acid (9 mL) and trifluoroacetic anhydride(1.53 mL, 10.85 mmol) was added NIS (2.569 g, 10.85 mmol) at roomtemperature. Then, the mixture was heated to 70-85° C. and was stirredfor 2 h. The reaction mixture was cooled to room temperature andsaturated sodium carbonate solution was added slowly to neutralize thesolution. Then, the aqueous mixture was extracted with ethyl acetate(2×50 mL). The combined extracts were washed successively with saturatedsodium thiosulfate solution (100 mL) and brine solution (100 mL) andwere dried over anhydrous sodium sulfate. Filtration of the drying agentand concentration of the solvent gave a crude product, which waspurified by silica gel chromatography on a Biotage (40 m) column toafford 1.02 g (45% yield) of1,4-dimethyl-3-iodo-6-(trifluoromethyl)-1H-pyridin-2-one as an amorphouswhite solid. ES-LRMS: m/z 318.1 (M+H), 381.2 (M+Na⁺ CH₃CN).f) Preparation ofN-[(1,1-dimethylethoxyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester

To a suspension of zinc dust (1.96 g, 30 mmol) in THF (1.0 mL) was added1,2-dibromoethane (172 μL, 2 mmol)) at room temperature. This suspensionwas heated to 60-65° C. with a heat gun until evolution of ethylene gasceased. Then, the suspension was cooled to room temperature andtrimethylchlorosilane (150 μL, 1.2 mmol)) was added and the mixture wasstirred for 15 min. A suspension of1,4-dimethyl-3-iodo-6-(trifluoromethyl)-2-pyridone (2.4 g, 7.57 mmol) inDMA (6 mL) was warmed with a heat gun to effect dissolution and wasadded in one portion to the reaction mixture. After addition, themixture was heated to 70-75° C. and was stirred for 2 h, at which timethe TLC analysis of an aliquot, which had been quenched with saturatedammonium chloride solution, indicated the absence of starting material.The reaction mixture was diluted with THF (5 mL), was cooled to roomtemperature and the excess zinc dust was allowed to settle.

The above prepared solution containing the zinc compound (7.57 mmol) wasadded to a solution of Pd(dba)₂ (274 mg, 0.478 mmol), trifurylphosphine(391 mg, 1.287 mmol) andN-[(1,1-dimethylethoxy)carbonyl]-4-iodo-L-phenylalanine methyl ester (3g, 7.411 mmol) in THF (7 mL) at room temperature and the light yellowmixture was stirred for 40 h at 50-55° C. The reaction mixture waspoured into a saturated ammonium chloride solution and was extractedwith ethyl acetate (3×70 mL). The combined extracts were washed withbrine solution (100 mL) and were dried over anhydrous magnesium sulfate.Filtration of the drying agent and concentration gave the crude product,which was purified by silica gel chromatography on a Biotage (40 m)column to obtain 1.289 g (36% yield) ofN-[(1,1-dimethylethoxyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester as an amorphous white solid. ES-HRMS m/e calcd forC₂₃H₂₇F₃N₂O₅ (M+Na) 491.1764. found 491.1764.g) Preparation of4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester hydrochloride salt

AN-[(1,1-dimethylethoxyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester (253 mg, 0.54 mmol) was treated with 4 N HCl solution indioxane (4.0 mL) at room temperature. The solution was stirred for 2 has a white solid was formed. The mixture was concentrated and theresidue was dissolved in methanol. After removal of methanol, theresidue was dried under high vacuum to obtain 221 mg (100% yield) of4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester hydrochloride salt as an amorphous white solid. ES-LRMS m/z369.3 (M+H), 410.3 (M+CH3CN).h) Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester

To a suspension of4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester hydrochloride salt (216 mg, 0.53 mmol) and2,6-dichlorobenzoyl chloride (120 mg, 0.57 mmol) in THF (6 mL) was addedDIEA (210 μL, 1.19 mmol) at room temperature. After 5 min, a clearsolution was obtained which was stirred for 18 h. Then, the mixture wasdiluted with ethyl acetate (50 mL). The ethyl acetate solution waswashed successively with 0.5 N HCl (50 mL), saturated NaHCO₃ solution(50 mL) and brine solution (50 mL) and was dried over anhydrousmagnesium sulfate. Filtration of the drying agent and concentration gavecrude product, which was dissolved in ethyl acetate (˜3 mL) and hexanes(˜3-4 mL) was added and stored in the refrigerator. The solid wascollected and washed with hexanes. After drying under high vacuum, 270mg (93.5% yield) ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester was obtained a white solid: mp 170-173° C. ES-HRMS m/ecalcd for C₂₅H₂₁Cl₂F₃N₂O₄ (M+Na) 563.0724. found 563.0730.Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl)-L-phenylalanine

To a suspension ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester (243 mg, 0.45 mmol) in ethanol (5 mL) was added 1N aqueoussodium hydroxide solution (1.8 mL) at room temperature. The mixture washeated to 45-50° C. and was stirred for 2 h. Then, the ethanol wasremoved under vacuum and the residue was diluted with water (20 mL). Theaqueous solution was washed with ethyl acetate (50 mL) to remove anyneutral impurities. The aqueous layer was acidified with 1.0 N HCl andthe product was extracted with ethyl acetate (2×50 mL). The combinedorganic extracts were washed with brine solution (50 mL) and were driedover anhydrous magnesium sulfate. Filtration of the drying agent andconcentration afforded 186 mg (79% yield) ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineas an amorphous white solid. ES-HRMS m/e calcd for C₂₄H₁₉Cl₂F₃N₂O₄(M+Na) 549.0567. found 549.0573.

Example 45 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl)-L-phenylalanine

N-[(2-chloro-6-methylphenyl)carbonyl]-4-(1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl)-L-phenylalaninewas prepared from4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalaninemethyl ester and 2-chloro-6-methylbenzoyl chloride using the generalprocedures described in example 44. ES-LRMS m/z 507.1 (M+H), 529.1(M+Na).

Example 46 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester

N-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester can be prepared fromN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineand iodoethane using the general procedure described in example 38.

Example 47 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester

N-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester can be prepared fromN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineand 2-[(N,N-diethyl)amino]ethyl chloride hydrochloride using the generalprocedure described in example 39.

Example 48 Preparation ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester

N-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester can be prepared fromN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineand 1-chloroethyl acetate using the general procedure described inexample 40.

Example 49 Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester

N-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester can be prepared fromN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineand iodoethane using the general procedure described in example 38.

Example 50 Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester

N-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester can be prepared fromN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineand 2-[(N,N-diethyl)amino]ethyl chloride hydrochloride using the generalprocedure described in example 39.

Example 51 Preparation ofN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester

N-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester can be prepared fromN-[(2,6-dichlorophenyl)carbonyl]-4-[11,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineand 1-chloroethyl acetate using the general procedure described inexample 40.

BIOASSAY EXAMPLES Example A

VLA-4/VCAM-1 Screening Assay

VLA-4 antagonist activity, defined as ability to compete for binding toimmobilized VCAM-1, was quantitated using a solid-phase, dual antibodyELISA. VLA-4 (α4β1 integrin) bound to VCAM-1 was detected by a complexof anti-integrin β1 antibody: HRP-conjugated anti-mouse IgG: chromogenicsubstrate (K-Blue). Initially, this entailed coating 96 well plates(Nunc Maxisorp) with recombinant human VCAM-1 (0.4 μg in 100 μl PBS),sealing each plate and then allowing the plates to stand at 4° C. for^(˜)18 hr. The VCAM-coated plates were subsequently blocked with 250 μLof 1% BSA/0.02% NaN₃ to reduce non-specific binding. On the day ofassay, all plates were washed twice with VCAM Assay Buffer (200 μl/wellof 50 mM Tris-HCl, 100 mM NaCl, 1 mM MnCl₂, 0.05% Tween 20; pH 7.4).Test compounds were dissolved in 100% DMSO and then diluted 1:20 in VCAMAssay Buffer supplemented with 1 mg/mL BSA (i.e., final DMSO=5%). Aseries of 1:4 dilutions were performed to achieve a concentration rangeof 0.005 nM-1.563 μM for each test compound. 100 μl per well of eachdilution was added to the VCAM-coated plates, followed by 10 μl of Ramoscell-derived VLA-4. These plates were sequentially mixed on a platformshaker for 1 min, incubated for 2 hr at 37° C., and then washed fourtimes with 200 μl/well VCAM Assay Buffer. 100 μl of mouse anti-humanintegrin β1 antibody was added to each well (0.6 μg/mL in VCAM AssayBuffer+1 mg/mL BSA) and allowed to incubate for 1 hr at 37° C. At theconclusion of this incubation period, all plates were washed four timeswith VCAM Assay Buffer (200 μl/well). A corresponding second antibody,HRP-conjugated goat anti-mouse IgG (100 μl per well @ 1:800 dilution inVCAM Assay Buffer+1 mg/mL BSA), was then added to each well, followed bya 1 hr incubation at room temperature and concluded by three washes (200μl/well) with VCAM Assay Buffer. Color development was initiated byaddition of 100 μl K-Blue per well (15 min incubation, room temp) andterminated by addition of 100 μl Red Stop Buffer per well. All plateswere then read in a UV/V is spectrophotometer at 650 nM. Results werecalculated as % inhibition of total binding (i.e., VLA-4+VCAM-1 in theabsence of test compound).

The results are provided in the following Table I (A=IC₅₀<1 nM,B=IC₅₀<10 nM):

TABLE I Compound of Example Activity in VCAM/VLA-4 ELISA Assay  8 A 10 B15 B 17 B 24 A 28 A

Example B

Ramos (VLA-4)/VCAM-1 Cell-Based Screening Assay Protocol

Materials:

Soluble recombinant human VCAM-1 (mixture of 5- and 7-Ig domain) waspurified from CHO cell culture media by immunoaffinity chromatographyand maintained in a solution containing 0.1 M Tris-glycine (pH 7.5), 0.1M NaCl, 5 mM EDTA, 1 mM PMSF, 0.02% 0.02% NaN₃ and 10 μg/mL leupeptin.Calcein-AM was purchased from Molecular Probes Inc.

Methods:

VLA-4 (α4β1 integrin) antagonist activity, defined as ability to competewith cell-surface VLA-4 for binding to immobilized VCAM-1, wasquantitated using a Ramos-VCAM-1 cell adhesion assay. Ramos cellsbearing cell-surface VLA-4, were labeled with a fluorescent dye(Calcein-AM) and allowed to bind VCAM-1 in the presence or absence oftest compounds. A reduction in fluorescence intensity associated withadherent cells (% inhibition) reflected competitive inhibition of VLA-4mediated cell adhesion by the test compound.

Initially, this entailed coating 96 well plates (Nunc Maxisorp) withrecombinant human VCAM-1 (100 ng in 100 μl PBS), sealing each plate andallowing the plates to stand at 4° C. for ≈18 hr. The VCAM-coated plateswere subsequently washed twice with 0.05% Tween-20 in PBS, and thenblocked for 1 hr (room temperature) with 200 μl of Blocking Buffer (1%BSA/0.02% thimerosal) to reduce non-specific binding. Following theincubation with Blocking Buffer, plates were inverted, blotted and theremaining buffer aspirated. Each plate was then washed with 300 μl PBS,inverted and the remaining PBS aspirated.

Test compounds were dissolved in 100% DMSO and then diluted 1:25 in VCAMCell Adhesion Assay Buffer (4 mM CaCl₂, 4 mM MgCl₂ in 50 mM TRIS-HCl, pH7.5) (final DMSO=4%). A series of eight 1:4 dilutions were performed foreach compound (general concentration range of 1 nM-12,500 nM). 100μl/well of each dilution was added to the VCAM-coated plates, followedby 100 μl of Ramos cells (200,000 cells/well in 1% BSA/PBS). Platescontaining test compounds and Ramos cells were allowed to incubate for45 min at room temperature, after which 165 μl/well PBS was added.Plates were inverted to remove non-adherent cells, blotted and 300μL/well PBS added. Plates were again inverted, blotted and the remainingbuffer gently aspirated. 100 μl Lysis Buffer (0.1% SDS in 50 mMTRIS-HCl, pH 8.5) was added to each well and agitated for 2 min on arotary shaking platform. The plates were then read for fluorescenceintensity on a Cytofluor 2300 (Millipore) fluorecence measurement system(excitation=485 nm, emission=530 nm). The results are shown in thefollowing table:

The results are provided in the following Table II (A=IC₅₀<100 nM,B=IC₅₀<10000 nM, C=IC₅₀<5,000 nM):

TABLE II Activity in VCAM/VLA-4 Ramos Cell Compound of Example Assay  8B 10 B 15 C 24 B 28 C 30 B 31 B 32 B 33 A 34 B 35 B 36 A 37 B

Example C

Alpha4-Beta7 Assay Protocol

Two weeks to one day prior to the assay, Nunc high-binding F96 Maxisorpimmuno plates, #442404 or #439454, were coated with 25 ng/well (0.25μg/ml) MadCAM in a volume of 100 μl/well. The plates were covered withsealer and wrapped in saran wrap followed by incubation in therefrigerator for at least 24 hours. The coating buffer employed was: 10mM carbonate/bicarbonate buffer made up from: 0.8 g/L sodium carbonateand 1.55 g/L sodium bicarbonate adjusted to pH 9.6 with 1 N HCl.

Assay buffers consisted of the following:

-   Wash Buffer: 0.05% Tween 20 in PBS-   Blocking Buffer: 1% Nonfat Dry Milk in PBS-   Labeling Buffer: PBS-   Cell Buffer: RPMI 1640 medium (no additives)-   Binding Buffer: 1.5 mM CaCl₂-   0.5 mM MnCl₂-   50 mM TRIS-HCl; add NaOH dropwise to pH 7.5-   Bring to volume in H₂O-   Adjust to pH 7.5-   Dilution Buffer: 4% DMSO in Binding Buffer

Plates were washed 2× with wash buffer and then blocked at roomtemperature for at least 1 hour with Blocking Buffer. Sealed plates weresometimes blocked overnight in the refrigerator. Plates were then washedwith PBS and hand blotted dry. Remaining liquid was aspirated from thewells.

Sufficient RPMI 8866 cells were removed from stock for assay (2×10⁶cells/ml×10 ml/plate×number of plates) and placed in a centrifuge tube.The tubes were filled to volume with PBS and were spun at 200×G for 8minutes. The buffer was poured off and the cells were resuspended to10×10⁶/ml in PBS and a stock solution of calcein in DMSO (5 mg/mL) wasadded at 5 μl/ml of cell suspension. The suspension was incubated for 30minutes at 37° C. in dark. The cells were then washed with PBS. The PBSwas poured off and the cells resuspended in cell buffer at aconcentration of 2×10⁶ cells/mL for plating in the assay.

Stock solution of test compounds at 25× first dilution desired in 100%DMSO were prepared. First dilutions for the standard, as well as testcompounds, were 1:25 into straight Binding Buffer, while the remainingserial dilutions were into Dilution Buffer (Binding Buffer/4% DMSO).Stock concentrations and dilutions of compounds for screening weredetermined by anticipated activity.

For the assay, 129 μl Binding Buffer was plated into first row of wellsand 100 μl Dilution Buffer was plated into remaining wells. A 5.4 μlaliquot of each compound was pipetted into appropriate, labeled wells,in triplicate. The compounds were next diluted down the plate (34 μl+100μl=>4-fold dilution). For controls, 100 μl of Dilution Buffer+100 μlCell Buffer were plated into the nonspecific background wells (no cells,no compound) and 100 μl Dilution Buffer+100 μl cells were plated intothe total binding wells (no compound=100% binding). Labeled cells at2×10⁶ cells/ml, 100μl/well (=2×10⁵ cells/well) were added to each wellcontaining compound. The plates were sealed and incubated in the darkfor 45 minutes at room temperature. Following incubation, unbound cellswere removed by adding 150 μl PBS/well. The plates were inverted,blotted onto paper towels and washed by gently adding 200 μl PBS towells and blotting again. Remaining buffer was carefully aspirated fromthe wells. A final 100 μl PBS was added to each well.

The plates were then read for fluorescence intensity on a Cytofluor 2300(Millipore) fluorecence measurement system (excitation=485 nm,emission=530 nm). IC₅₀s of each compound were determined by linearregression analysis.

The results are shown in the following table:

The results are provided in the following Table III:

TABLE III Activity in MadCAM/RPMI Cell Assay Compound (A = IC₅₀ < 100nM, of Example B = IC₅₀ < 10000 nM, C = IC₅₀ < 5,000 nM) 30 B 31 B 32 B33 A 34 B 35 C 36 B 37 B

1. A compound of the formula:

wherein R₁ is a group of the formula Y-1

wherein R₂₂ and R₂₃ are independently hydrogen, lower alkyl, loweralkoxy, cycloalkyl, aryl, arylalkyl, nitro, cyano, lower alkylthio,lower alkylsulfinyl, lower alkyl sulfonyl, lower alkanoyl, halogen, orperfluorolower alkyl and at least one of R₂₂ and R₂₃ is other thanhydrogen, and R₂₄ is hydrogen, lower alkyl, lower alkoxy, aryl, nitro,cyano, lower alkyl sulfonyl, or halogen; or R₁ is a a group of theformula Y-2, which is a five or six membered heteroaromatic ring bondedvia a carbon atom to the amide carbonyl wherein said ring contains one,two or three heteroatoms selected from the group consisting of N, O andS and one or two atoms of said ring are independently substituted bylower alkyl, cycloalkyl, halogen, cyano, perfluoro lower alkyl, or aryland at least one of said substituted atoms is adjacent to the carbonatom bonded to the amide carbonyl; or R₁ is a group of the formula Y-3which is a 3-7 membered ring of the formula:

wherein R₂₅ is lower alkyl, unsubstituted or fluorine substituted loweralkenyl, or a group of formula R₂₆—(CH₂)_(e)—, R₂₆ is aryl, heteroaryl,azido, cyano, hydroxy, lower alkoxy, lower alkoxycarbonyl, loweralkanoyl, lower alkylthio, lower alkyl sulfonyl, lower alkyl sulfinyl,perfluoro lower alkanoyl, nitro, or R₂₆ is a group of formula —NR₂₈R₂₉,wherein R₂₈ is hydrogen or lower alkyl, R₂₉ is hydrogen, lower alkyl,lower alkoxycarbonyl, lower alkanoyl, aroyl, perfluoro loweralkanoylamino, lower alkyl sulfonyl, lower alkylaminocarbonyl,arylaminocarbonyl; or R₂₈ and R₂₉, taken together with the attachednitrogen atom, form a 4, 5 or 6-membered saturated heterocyclic ringoptionally containing one additional heteroatom selected from O, S, andN—R₄₀, Q is —(CH₂)_(f)O—, —(CH₂)_(f)S—, —(CH₂)_(f)N(R₂₇)—, —(CH₂)_(f)—,R₂₇ is H, lower alkyl, aryl, lower alkanoyl, aroyl or loweralkoxycarbonyl, R₄₀ is H, lower alkyl, aryl, lower alkanoyl, aroyl orlower alkoxycarbonyl, the carbon atoms in the ring are unsubstituted orsubstituted by lower alkyl or halogen, e is an integer from 0 to 4, andf is an integer from 0 to 3; R₂ is hydrogen, lower alkyl, substitutedlower alkyl, aryl, or aryl lower alkyl; R₃ is hydrogen, halogen, loweralkyl, trifluoromethyl, or aryl; R₄ is hydrogen, halogen, lower alkyl,or aryl; R₅ is hydrogen, lower alkyl, lower alkoxy, or trifluoromethyl,or OH; R₆ is hydrogen, lower alkyl, lower alkylcarbonyloxy lower alkyl,or R₆ is a group of formula P-3:

wherein: R₃₂ is hydrogen or lower alkyl; R₃₃ is hydrogen, lower alkyl,aryl; R₃₄ is hydrogen or lower alkyl; h is an integer from 0 to 2; g isan integer from 0 to 2; the sum of h and g is 1 to 3; or R₆ is a groupof formula P-4:

wherein: R₃₂, g, and h are as previously defined; Q′ is O, S,—(CH₂)_(j)—, or a group of the formula N—R₃₅; wherein R₃₅ is hydrogen,lower alkyl, lower alkanoyl, lower alkoxycarbonyl; j is 0, 1 or 2; andR₇ is hydrogen, chloro, lower alkoxy, or lower alkyl; or thepharmaceutically acceptable salts thereof.
 2. A compound of claim 1wherein Q is —(CH₂)_(f)O—, —(CH₂)_(f)S—, or, —(CH₂)_(f)—.
 3. A compoundof claim 2 wherein R₅ is hydrogen, lower alkyl, or trifluoromethyl.
 4. Acompound of claim 1 wherein R₁ is a group of the formula Y-1 wherein R₂₂and R₂₃ are independently lower alkyl or halogen and R₂₄ is hydrogen. 5.A compound of claim 1 wherein R₁ is a group of the formula Y-1 whereinR₂₂ and R₂₃ are independently hydrogen, lower alkyl or halogen and R₂₄is hydrogen, lower alkyl or lower alkoxy.
 6. A compound of claim 5wherein R₂₂ and R₂₃ are independently hydrogen, lower alkyl or halogen,R₂₄ is hydrogen or lower alkoxy and R₂ is aryl lower alkyl.
 7. Acompound of claim 1 wherein R₁ is a group of the formula Y-3 wherein R₂₅a group of formula R₂₆—(CH₂)_(e)—, wherein R₂₆ is lower alkoxy, Q is—(CH₂)_(f)—, e is an integer from 0 to 4 and f is an integer from 0 to3.
 8. A compound of claim 1 wherein R₁ is a group of the formula Y-3 andR₂ is hydrogen, lower alkyl, substituted lower alkyl or aryl loweralkyl.
 9. A compound of claim 8 wherein R₁ is a group of the formula Y-3wherein R₂₅ is a group of formula R₂₆—(CH₂)_(e)—, wherein R₂₆ is loweralkoxy, Q is —(CH₂)_(f)—, e is an integer from 0 to 4, f is an integerfrom 0 to 3; and R₂ is aryl lower alkyl.
 10. A compound of claim 1wherein R₂ is hydrogen, lower alkyl or aryl lower alkyl; R₃ is hydrogen;R₄ is hydrogen or halogen; R₅ is hydrogen, lower alkyl, ortrifluoromethyl, or lower alkoxy; R₆ is hydrogen or lower alkyl; and R₇is hydrogen, chloro, lower alkoxy, or lower alkyl.
 11. A compound ofclaim 10 wherein R₁ is a group of the formula Y-1.
 12. A compound ofclaim 11 wherein R₁ is a group of the formula Y-1 wherein R₂₂ and R₂₃are independently lower alkyl or halogen and R₂₄ is hydrogen or loweralkoxy.
 13. A compound of claim 11 wherein R₁ is a group of the formulaY-1 wherein R₂₂ and R₂₃ are independently hydrogen, lower alkyl orhalogen and R₂₄ is hydrogen, lower alkyl or lower alkoxy.
 14. A compoundof claim 13 wherein R₁ is a group of the formula Y-1 wherein R₂₂ and R₂₃are independently hydrogen, lower alkyl or halogen, R₂₄ is hydrogen andR₂ is aryl lower alkyl.
 15. A compound of claim 10 wherein R₁ is a groupof the formula Y-2.
 16. A compound of claim 10 wherein R₁ is a group ofthe formula Y-3.
 17. A compound of claim 16 wherein R₁ is a group of theformula Y-3 wherein R₂₅ is a group of formula R₂₆—(CH₂)_(e)—, whereinR₂₆ is lower alkoxy, Q is —(CH₂)_(f)—, e is an integer from 0 to 4 and fis an integer from 0 to
 3. 18. A compound of claim 10 wherein R₁ is agroup of the formula Y-3 wherein R₂₅ is a group of formulaR₂₆—(CH₂)_(e)—, wherein R₂₆ is lower alkoxy, Q is —(CH₂)_(f)—, e is aninteger from 0 to 4, f is an integer from 0 to 3, and R₂ is aryl loweralkyl.
 19. A compound of claim 10 wherein R₂ is hydrogen, lower alkyl oraryl lower alkyl; R₃ is hydrogen; R₄ is halogen; R₅ is hydrogen; R₆ ishydrogen or lower alkyl; and R₇ is hydrogen, chloro, lower alkoxy, orlower alkyl.
 20. A compound of claim 3 wherein R₁ is a group of formulaY-1 or a group of formula Y-3; R₂ is hydrogen, lower alkyl or aryl loweralkyl; R₃ is hydrogen, lower alkyl, or trifluoromethyl; R₄ is hydrogenor halogen; R₅ is hydrogen, lower alkyl, or trifluoromethyl; R₆ ishydrogen, lower alkyl, lower alkylcarbonyloxy lower alkyl, a group offormula P-3; or a group of formula P-4, and R₇ is hydrogen or loweralkyl.
 21. A compound of claim 20 wherein R₂ is lower alkyl or aryllower alkyl; R₆ is hydrogen, lower alkyl, lower alkylcarbonyloxy loweralkyl, or a group of formula P-3; and R₇ is hydrogen.
 22. A compound ofclaim 21 wherein R₂₂ and R₂₃ are hydrogen, halogen or lower alkyl, R₂₄is hydrogen or lower alkoxy, R₂₅ is a group of formula R₂₆—(CH₂)_(e)—,wherein R₂₆ is lower alkoxy, Q is —(CH₂)_(f)—, e is an integer from 0 to4 and f is an integer from 0 to
 3. 23. A compound of claim 22 wherein R₂is lower alkyl, R₄ is hydrogen, and R₃ and R₅ are lower alkyl ortrifluoromethyl.
 24. A compound of claim 23 where R₆ is hydrogen.
 25. Acompound of claim 24 wherein R₁ is Y-3.
 26. A compound of claim 25 whichisN-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine.27. A compound of claim 23 wherein R₆ is lower alkyl.
 28. A compound ofclaim 27 which isN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester.
 29. A compound of claim 27 which isN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester.
 30. A compound of claim 27 which isN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester.
 31. A compound of claim 27 which isN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanineethyl ester.
 32. A compound of claim 23 wherein R₆ is loweralkylcarbonyloxy lower alkyl.
 33. A compound of claim 32 which isN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester.
 34. A compound of claim 32 which isN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester.
 35. A compound of claim 32 which isN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester.)
 36. A compound of claim 32 which isN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine1-(acetoxy)ethyl ester.
 37. A compound of claim 23 wherein R₆ is a groupof formula P-3 wherein R₃₂ is hydrogen; R₃₃ and R₃₄ are lower alkyl; oneof g and h is 1 and the other is
 0. 38. A compound of claim 37 which isN-[(2,6-dichlorophenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester.
 39. A compound of claim 37 which isN-[(2,6-dichlorophenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester.
 40. A compound of claim 37 which isN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,6-dimethyl-4-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester.
 41. A compound of claim 37 which isN-[(2-chloro-6-methylphenyl)carbonyl]-4-[1,4-dimethyl-6-(trifluoromethyl)-2-oxo-3-pyridinyl]-L-phenylalanine2-[(N,N-diethyl)amino]ethyl ester.
 42. A compound of claim 22 wherein R₆is hydrogen.
 43. A compound of claim 42 wherein R₂ is lower alkyl; R₃ ishydrogen; R₄ is hydrogen or halogen; and R₅ is hydrogen or lower alkyl.44. A compound of claim 43 wherein R₁ is Y-1.
 45. A compound of claim 44wherein R₂₂ and R₂₃ are lower alkyl or halogen, and R₂₄ is hydrogen. 46.A compound of claim 45 which isN-[(2-chloro-6-methylphenyl)carbonyl]-4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine.47. A compound of claim 45 which isN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine.48. A compound of claim 45 which isN-[(2-chloro-6-methylphenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalanine.49. A compound of claim 44 wherein R₂₂ and R₂₃ are halogen, and R₂₄ ishydrogen.
 50. A compound of claim 49 which isN-[(2,6-dichlorophenyl)carbonyl]-4-(1,4-dimethyl-2-oxo-3-pyridinyl)-L-phenylalanine.51. A compound of claim 44 wherein R₂₂ and R₂₃ are halogen or hydrogenand R₂₄ is lower alkoxy.
 52. A compound of claim 51 which isN-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine.53. A compound of claim 51 which isN-[(2-bromo-5-methoxyphenyl)carbonyl]-4-(5-bromo-1-methyl-2-oxo-3-pyridinyl)-L-phenylalanine.54. A compound of claim 43 wherein R₁ is Y-3.
 55. A compound of claim 54which is4-(5-chloro-1-methyl-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine.56. A compound of claim 21 wherein R₂₂ and R₂₃ are halogen or loweralkyl, R₂₄ is hydrogen or lower alkoxy, R₂₅ is a group of formulaR₂₆—(CH₂)_(e)—, wherein R₂₆ is lower alkoxy, Q is —(CH₂)_(f)—, e is aninteger from 0 to 4 and f is an integer from 0 to
 3. 57. A compound ofclaim 56 wherein R₂ is aryl lower alkyl; R₃ is hydrogen, R₄ is halogen,and R₅, R₆ and R₇ are hydrogen.
 58. A compound of claim 57 wherein R₁ isY-1.
 59. A compound of claim 58 which is4-(1-Benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[(2-chloro-6-methylphenyl)carbonyl]-L-phenylalanine.60. A compound of claim 57 wherein R₁ is Y-3.
 61. A compound of claim 60which is4-(1-Benzyl-5-chloro-2-oxo-3-pyridinyl)-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine.